CN110156779A - Inhibitors of influenza viruses replication - Google Patents

Abstract

The polymorphic of compound (1) or its pharmaceutically acceptable salt is the HCl salt 1/2H of compound (1) wherein being expressed as the compound (1) of structural formula₂The HCl salt 3H of the form A of O, compound (1)₂The form D of O, the form D of the HCl salt of compound (1), the form A of compound (1) and compound (1) toluene fulfonate form A.Amount of this kind of polymorphic for treating the influenza in biological sample or subject, its influenza virus being inhibited to replicate or reduce its influenza virus.

Description

Inhibitors of influenza viruses replication

The application be the applying date be on November 12nd, 2014, application No. is 201480071165.6 (PCT/US2014/ 065114) divisional application of the Chinese invention patent application of, entitled " inhibitors of influenza viruses replication ".

The cross reference of related application

This PCT application requires the equity of the U.S. Provisional Application No.61/903,572 submitted on November 13rd, 2013.This is right It is incorporated herein by reference in the form of its is complete than file.

Technical field

The present invention relates to the seriousness of the influenza infection for inhibiting the influenza virus of patient to replicate, treating or mitigate patient And the compound and solid form of the disease incidence of the influenza infection of preventative prevention or reduction patient.

Background technique

Influenza is propagated in the whole world in seasonal epidemics, cause annual hundreds of thousands of them it is dead-in pandemic disease year be Millions of people.For example, 3 flu outbreaks have occurred 20th century, causes tens of millions of people dead, be very popular every time due in people The appearance of novel strain in class.In general, propagation of these novel strains from existing influenza virus from other animal species to the mankind Cause.

The virulent big droplet of load that influenza generates when mainly coughing or sneeze by the infected is passing between men It broadcasts；Then these big droplets can rest on the mucous membrane of the upper respiratory tract of the susceptible individual close to (for example, in about 6 feet) the infected Surface.Propagating can also be and with directly or indirectly contacting for respiratory secretions, such as touches and polluted by influenza virus Surface, then touch eyes, nose or mouth.Adult can about 5 days will stream after 1 day or symptom start before there is symptom Sense is broadcast to other people.Child and the weak people of immune function may have infectiousness in 10 days or more after paresthesia epilepsy.

Influenza virus is the RNA virus of Orthomyxoviridae family (Orthomyxoviridae) comprising 5 categories: A type influenza Virus, Type B influenza virus, c-type influenza virus, ISA virus and the high soil of support are viral (Thogoto virus).

Influenza A category has 1 species, influenza A.Wild aquatic bird is the natural host of a large amount of A type influenzas.Have When, viral transmission to other species and it can then cause crushing outburst in poultry or Human Influenza is caused to be very popular.3 kinds In influenza type, A type virus is most toxic human pathogen and the disease that will cause most serious.According to these viruses Antibody response, influenza A can be subdivided into different serotypes.With the known mankind be very popular death toll sequence have confirmed that Human serotypes are as follows: H1N1 (causing spanish influenza in 1918), H2N2 (nineteen fifty-seven causes Asia influenza), H3N2 (nineteen sixty-eight Cause Mao flu), H5N1 (threat of being very popular in 2007-2008 influenza season), H7N7 (have rare zoonosis latent Can), H1N2 (endemic conditions in the mankind and pig), H9N2, H7N2, H7N3 and H10N7.

Type B Influenza Virus has 1 species, Type B influenza virus.Type B influenza almost specially infect the mankind and with A type influenza It compares uncommon.Other known unique animal vulnerable to Type B influenza infection is sea dog.Such influenza is according to than A type Slow 2-3 times of rate is mutated and therefore genetic diversity is low, only a kind of Type B anti-influenza sera type.Due to this antigen multiplicity The shortage of property, is usually obtaining a degree of Type B influenza immunity in one's early years.However, the mutation of Type B influenza is enough to make to hold It is immune long.Antigen change rate is reduced in this way, merges its limited host's variation (inhibiting across species antigenic shifts), it is ensured that Bu Huifa Raw Type B flu outbreak.

C-type Influenza Virus has 1 species, c-type influenza virus, infect the mankind and pig and can cause serious disease and Endemic conditions disease.However, c-type influenza is not common compared with other type influenzas and usually seems to cause that children's is slight Disease.

A, B and c-type influenza virus are closely similar in structure.Virion diameter is 80-120nm and usually approximate ball Body, although being likely to occur filamentous form.Unusual for virus, genome is not the nucleic acid of single segment； On the contrary, justice RNA is born in the segmentation that genome contains 7 or 8 segments.A type influenza gene group encodes 11 kinds of protein: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), M1, M2, NS1, NS2 (NEP), PA, PB1, PB1-F2 and PB2.

HA and NA is the macromolecular glycoprotein outside virion.HA is mediate retroviral combination target cell and viral genome Into the agglutinin of target cell, and NA is related to discharging filial generation from infection cell by cracking the sugar in conjunction with mature virion Virus.Therefore, these protein have become the target of antiviral drugs.Moreover, these protein are the antigen that can produce antibody. Influenza A is divided into hypotype according to the antibody response to HA and NA, formed H and N difference in such as H5N1 basis (referring to Above).

The indirect cost of direct cost and precautionary measures can be generated due to losing productivity and related medical influenza.In beauty State, influenza be the reason of causing per year over 10000000000 dollars of totle drilling costs, and according to estimates following pandemic disease can cause it is hundreds billion of The directly or indirectly cost of dollar.Prevention cost is also very high.It is possible that countries in the world government, which has taken multi-million dollar, H5N1 bird flu, which is very popular, to be prepared and plans, and cost is managed with purchase drug and vaccine and the rehearsal of development disaster and raising border The strategy of system is related.

Current treatment of influenza selection includes vaccine inoculation and carries out chemotherapy or chemoprophylaxis with antiviral drugs.Often Often to high risk group, such as children and the elderly, or the influenza for thering is asthma, diabetes or cardiopathic people to recommend inoculation anti influenza Vaccine.However, it is possible to through inoculation but still obtain influenza.Each season prepares the vaccine of some specific influenza strains again, but can not All bacterial strains including season active infections people in the world.Manufacturer with preparing and produce processing seasonal epidemics for about 6 months Required millions of doses；Sometimes, new or ignored bacterial strain becomes significant and infection population in the meantime, although these Crowd's vaccine inoculation (such as 2003-2004 influenza season H3N2 Fujian influenza).It may also be just infected simultaneously before inoculation Just infection assumes the bacterial strain of vaccine prevention, because vaccine needs about several weeks just to work.

In addition, the effect of these influenza vaccines, is variable.Due to the high mutation rate of virus, specific influenza vaccines are generally given to not Super protection in a few years.Due to virus quickly variation at any time, and different strains become advantage, and the vaccine prepared for certain year can It can be invalid in next year.

Similarly, since lacking RNA proofreading enzyme, the RNA polymerase of the RNA dependence of influenza vRNA is every about 10,000 Nucleotide (this is the approximate length of influenza vRNA) generates a nucleotides inserted mistake.Therefore, almost every kind of brand-new influenza is sick Poison is mutation-antigenic drift.If more than one virus stock has infected individual cells, genome is separated into 8 individually VRNA segment makes vRNA mix or be reconfigured.Quick variation on generated viral genetics generates antigenic shift and makes virus Infection new host species simultaneously overcome rapidly protective immunity.

Antiviral drugs can also be used for treatment influenza, and wherein neuraminidase inhibitor is particularly effective, but virus can be to mark Quasi- antiviral drugs develops drug resistance.This kind of activating agent can be prepared, so as to a variety of different chemical species, including chemistry Derivative or salt, or with different physical forms.Such as.They can have different polymorphics to be unbodied Object, or can exist with different solvations or hydrated state.By changing form, its physical characteristic can be changed.It is this kind of not Same form has different characteristics, especially as oral preparation.In particular, it is desirable to identify changing for the characteristic for showing improvement Into form, such as increased water-soluble and stability, better pharmaceutical preparation processability or the group for preparing row and oral administration The bioavilability for closing object increases.This kind of improved characteristic discussed can be changed in a manner of being beneficial to particular treatment effect Become.

The variation of the form of antivirotic can be one of many modes, wherein adjusting the physical characteristic of this kind of antivirotic It is more useful in treatment influenza.

Summary of the invention

Present invention relates generally to the polymorphic of compound (1) or its pharmaceutically acceptable salt, its is pharmaceutically acceptable Preparation, prepare this kind of compound polymorphous method and this kind of polymorphic inhibit influenza virus duplication, reduce influenza disease Purposes in the amount and treatment influenza of poison.

In one embodiment, the present invention relates to compound (1) or the polymorphics of its pharmaceutically acceptable salt, wherein Compound (1) is expressed as structural formula:

And wherein the polymorphic is selected from: the HCl salt 1/2H of compound (1)₂The form A of O；The HCl salt of compound (1) 3H₂The form D of O；The form D of the HCl salt of compound (1)；The A of compound (1)；With the shape of the toluene fulfonate of compound (1) Formula A.

In another embodiment, the present invention relates to pharmaceutically acceptable preparations, and it includes chemical combination disclosed herein The polymorphic or its pharmaceutically acceptable salt of object (1) and at least one pharmaceutically acceptable carrier or excipient.

In another embodiment, the present invention relates to the influenza virus inhibited in external biological sample or subject duplications Method.This method include compound (1) disclosed herein a effective amount of to the sample administration polymorphic or its pharmaceutically Acceptable salt.

In another embodiment, the present invention relates to the amounts for reducing the influenza virus in external biological sample or subject Method.This method include compound (1) disclosed herein a effective amount of to the sample administration polymorphic or its pharmaceutically Acceptable salt.

In another embodiment, the present invention relates to the methods of the influenza for the treatment of patient.This method includes to the sample Product apply a effective amount of compound (1) disclosed herein or the polymorphic of its pharmaceutically acceptable salt.

In another embodiment, the present invention relates to the HCl salt 1/2H of prepare compound (1)₂The side of the form A of O Method.This method includes mixing HCl and compound (1) in the solvent system for including water and one or more organic solvents, wherein The solvent system has the water activity of 0.05-0.85.Compound (1) can be solvation or non-solvated and/or nothing It is setting or crystallization.

In another embodiment, the present invention relates to the HCl salt 3H of prepare compound (1)₂The method of the form D of O. This method comprises: mixing HCl and compound in the solvent system including water or including water and one or more organic solvents (1), wherein the solvent system has the water activity equal to or more than 0.9, such as 0.9-1.0；Or including water or including water With the HCl salt 1/2H for stirring compound (1) in the solvent system of one or more organic solvents₂The form A of O, wherein described Solvent system decompression is equal to or more than 0.9 water activity, such as 0.9-1.0.Compound (1) can be solvation or non-solvent It is changing and/or unbodied or crystallization.

In another embodiment, the present invention relates to the methods of the form D of the HCl salt of prepare compound (1).This method HCl salt 1/2H including making compound (1)₂The form A of O is dehydrated.

In another embodiment, the present invention relates to the methods of the form A of prepare compound (1).This method is included in The solvate of amorphous compound (1) or compound (1) is stirred in solvent system including water and ethyl alcohol.

In another embodiment, the present invention relates to the methods of the form A of the toluene fulfonate of prepare compound (1). This method includes stirring the solvate of amorphous compound (1) or compound (1), p- toluenesulfonic acid and including the molten of acetonitrile The mixture of agent system.

The 2- methyl THF solvate of compound (1) is also covered in the present invention.

In another embodiment, the present invention relates to the influenza virus amounts reduced in external biological sample or subject Method, including the polymorphic to a effective amount of compound (1) disclosed herein of the sample administration.

In another embodiment, the present invention relates to the influenza virus duplications for inhibiting external biological sample or subject Method, including the polymorphic to a effective amount of compound (1) disclosed herein of the sample administration.

In another embodiment, the present invention relates to the methods of the influenza for the treatment of subject, including apply to the subject With the polymorphic of the compound disclosed herein (1) of therapeutically effective amount.

The invention also includes the polymorphics of compound disclosed herein (1) in the influenza virus duplication for inhibiting subject Purposes, in the purposes in the influenza virus amount for reducing subject or the purposes in the influenza for treating subject.The invention also includes The polymorphic of compound (1) disclosed herein is in preparation for inhibiting the influenza virus of subject to replicate, reducing the stream of subject Purposes in the medicament of the influenza of the amount or treatment subject of Influenza Virus.

On the other hand, the present invention relates to compound (1) or its pharmaceutically acceptable salt (such as compounds (1) HCl salt 1/2H₂The HCl salt 3H of the form A of O, compound (1)₂The form D of O, the form D of the HCl salt of compound (1), change Close object (1) form A and compound (1) toluene fulfonate form A) dosage, be 100mg-1,600mg.

Detailed description of the invention

Fig. 1 and 2 is the HCl salt 1/2H of compound (1) respectively₂X-ray powder diffraction (XRPD) pattern of the form A of O And C¹³Solid state NMR spectroscopy (C¹³SSNMR spectrum).

Fig. 3 and 4 is the HCl salt 3H of compound (1) respectively₂The XRPD pattern and C of the form D of O¹³The spectrum of SSNMR.

Figures 5 and 6 are the XRPD pattern and C of the form D of the HCl salt of compound (1) respectively¹³The spectrum of SSNMR.

Fig. 7 and 8 is the XRPD pattern and C of the form A of compound (1) respectively¹³The spectrum of SSNMR.

Fig. 9 is the XRPD pattern of the form A of the toluene fulfonate of compound (1).

Figure 10 is the XRPD pattern of 2- methyltetrahydrofuran (2-MeTHF) solvate of compound (1).

Figure 11 is the XRPD pattern of the amorphous form of compound (1).

Figure 12 be in variation between the different polymorphs of the HCl salt of compound (1) temperature to the phasor of water activity.

Figure 13 is the HCl salt 1/2H for showing 1200mg/600mg compound (1)₂The work of the form A dosage group of O in people The AUC virus being attenuated in influenza challenge model reduces the schematic diagram of (shedding).

Detailed description of the invention

I. solid form

It is expressed as the compound (1) of structural formula:

And its pharmaceutically acceptable salt can be used for that influenza virus is inhibited to replicate and be also described in WO 2010/ In 148197.

Compound (1), which can be used as different polymorphic forms, to be existed.As this field knows, polymorphism is chemical combination The object crystallization different as more than one or " polymorphic " plant paracrystalline ability.Polymorph is that have at least two different rows The polymorphic of the compound molecule of the solid crystal phase or solid-state form of the compound of column.The polymorphic for being arbitrarily designated compound can To be defined as identical molecular formula or form, but the crystalline texture of two kinds of different chemical compounds is different in terms of chemical structure. Generally, different polymorphs, such as X-ray powder diffraction (XRPD) pattern, thermogravimetric analysis can be characterized with analysis method (TGA) and differential scanning calorimetry (DSC)；Or different polymorphics is characterized according to its fusing point or other techniques known in the art Object.The term as used herein " polymorphic " includes solvate and the net polymorphic without any solvate.

" compound (1) " used herein refers to the free alkali form of compound (1).Therefore, " the HCl of compound (1) Salt the HCl salt of the free alkali compound " refer to and " compound (1) toluene fulfonate ".Note that compound (1) and compound (1) salt can be it is solvation or non-solvated, except otherwise indicated.Furthermore, it is noted that compound (1) and compound (1) salt can be it is crystallization or unbodied, except otherwise indicated.

In one embodiment, the present invention relates to the HCl salt 1/2H of compound (1)₂The polymorphic A of O.The form is The polymorphic of the HCl salt of compound (1) comprising the water as semi-normal in each compound (1) in solvate.One In a specific embodiment, the HCl salt 1/2H of compound (1)₂The form A of O is characterized in that being equivalent to X-ray powder One or more peaks of 10.5,5.2,7.4 and 18.9 (± 0.2 degree) 2- θ values of measurement are spent in last diffraction pattern.At another In specific embodiment, the 1/2H of the HCl salt of compound (1)₂O form A, which is further characterized in that, is being equivalent to X-ray powder One or more that 25.2 ± 0.2,16.5 ± 0.2,18.1 ± 0.2 and 23.0 ± 0.22- θ value of measurement is spent in last diffraction pattern A peak.In another specific embodiment, the HCl salt 1/2H of compound (1)₂The form A of O is characterized by having XRPD pattern has the characteristic peak indicated at the position listed in such as the following table 2 with θ ± 0.2 2-.It is specific real at another It applies in scheme, the HCl salt 1/2H of compound (1)₂The form A of O is characterized by having substantially identical to those shown in Fig. 1 XRPD pattern.XRPD pattern is obtained using Cu K alpha ray at room temperature.In another specific embodiment, compound (1) HCl salt 1/2H₂The polymorphic A of O is characterized in that in C¹³In SSNMR spectrum 29.2,107.0,114.0 and 150.7 (± 0.3ppm) place has one or more features peak.In another specific embodiment, the HCl salt 1/ of compound (1) 2H₂The polymorphic A of O is further characterized in that in C¹³Have at 22.1,24.6,47.7 and 54.8 (± 0.3ppm) in SSNMR spectrum There is one or more features peak.In another specific embodiment, the HCl salt 1/2H of compound (1)₂The form A's of O It is characterized by the C listed in table 3¹³The peak SSNMR.In another specific embodiment, the HCl salt 1/ of compound (1) 2H₂The form A of O be characterized by substantially with identical C shown in Fig. 2¹³SSNMR spectrum.

In another embodiment, the present invention relates to the HCl salt 3H of compound (1)₂The polymorphic F of O.This form It is the polymorphic of the HCl salt of compound (1) comprising the water as (1) 3 equivalent of compound each in solvate.At one In specific embodiment, the HCl salt 3H of compound (1)₂The form D of O is characterized in that being equivalent to spreads out in X-ray powder It penetrates in pattern and spends one or more peaks of the 2- θ value of 7.1,11.9,19.2 and 12.4 (± 0.2) of measurement.In another tool In the embodiment of body, the HCl salt 3H of compound (1)₂The form D of O is further characterized in that be equivalent to spreads out in X-ray powder It penetrates in pattern and spends one or more peaks of 16.4,21.8 and 23.9 (± 0.2) 2- θ values of measurement.It is specific real at another It applies in scheme, the HCl salt 3H of compound (1)₂The form D of O is characterized by XRPD pattern, has in such as the following table 5 In 2- θ ± 0.2 is indicated at the position listed characteristic peak.In another specific embodiment, the HCl of compound (1) Salt 3H₂The form D of O is characterized by XRPD pattern substantially identical with Fig. 3.XRPD pattern uses CuK α at room temperature Ray obtains.In another specific embodiment, the HCl salt 3H of compound (1)₂The polymorphic F of O is characterized in that C¹³Peak in SSNMR spectrum at 20.7,27.4,104.8,142.5,178.6 (± 0.3ppm).In another specific implementation In scheme, the HCl salt 3H of compound (1)₂The polymorphic F of O, which is further characterized in that, to be equivalent in C¹³154.3 in SSNMR spectrum, 20.3, one or more peaks of 132.3 and 21.1 (± 0.3ppm).In another specific embodiment, compound (1) HCl salt 3H₂The form D of O is characterized by the C listed in table 6¹³The peak SSNMR.In another specific embodiment, The HCl salt 3H of compound (1)₂The form D of O be characterized by substantially with identical C shown in Fig. 4¹³SSNMR spectrum.

In another embodiment, the present invention relates to the polymorphic D of the HCl salt of compound (1).This form is chemical combination The non-solvent form of the HCl salt of object (1).In a specific embodiment, the form D's of the HCl salt of compound (1) Be characterized in that in X-ray powder diffraction figure case 5.8,17.1 and 19.5 (± 0.2) degree one of 2- θ value to spend measurement or Multiple peaks.In another specific embodiment, the form D of the HCl salt of compound (1) is characterized in that in X-ray powder One or more peaks of 5.3,10.5 and 15.9 (± 0.2) degree 2- θ value of measurement are spent in diffraction pattern.It is specific at another In embodiment, the form D of the HCl salt of compound (1) is characterized by XRPD pattern, with the position listed in table 7 Sentence the characteristic peak of the expression of 2- θ ± 0.2.In another specific embodiment, the spy of the form D of the HCl salt of compound (1) Sign is with XRPD pattern substantially same as shown in Figure 5.XRPD pattern is obtained using Cu K alpha ray.It is specific at another Embodiment in, the form D of the HCl salt of compound (1) is characterized by C¹³In SSNMR spectrum 29.4,53.4, 113.3, the peak at 135.4,177.8 (± 0.3ppm).In another specific embodiment, the HCl salt shape of compound (1) Formula D, which is further characterized in that, to be equivalent in C¹³One in SSNMR spectrum at 22.9,23.9,26.0 and 31.6 (± 0.3ppm) Or multiple peaks.In another specific embodiment, the form D of the HCl salt of compound (1), which is characterized by table 8, arranges C out¹³The peak SSNMR.In another specific embodiment, the form D of the HCl salt of compound (1) is characterized by base C identical with Fig. 6 in sheet¹³SSNMR spectrum.

In one embodiment, the present invention relates to the polymorphic A of compound (1).This form is the trip of compound (1) Nonsolvated forms from alkali.In a specific embodiment, the feature of the form A of compound (1) is to be equivalent in X- One or more peaks of 15.5,18.9 and 22.0 (± 0.2) 2- θ values of measurement are spent in powder diffraction pattern.Another In a specific embodiment, the form A of compound (1), which is further characterized in that, to be equivalent in X-ray powder diffraction figure case to spend One or more peaks of 11.8,16.9,25.5 and 9.1 (± 0.2) 2- θ values of measurement.In another specific embodiment, The form A of compound (1) is characterized by XRPD pattern, and having is indicated on the position listed in table 10 with θ ± 0.2 2- Characteristic peak.In another specific embodiment, the form A of compound (1) be characterized by substantially with institute in Fig. 7 Show identical XRPD pattern.XRPD pattern is obtained using Cu K alpha ray at room temperature.In another specific embodiment, The form A of compound (1) is characterized by C¹³In 21.0,28.5,50.4,120.8,138.5 and in SSNMR spectrum Peak at 176.2 (± 0.3ppm).In another specific embodiment, the form A of compound (1) is characterized by C¹³Peak in SSNMR spectrum at 30.1,25.9,22.8 and 25.0 (± 0.3ppm).In another specific embodiment, The form A of compound (1) is characterized by the C listed in table 11¹³The peak SSNMR.In another specific embodiment, change Close object (1) form A be characterized by substantially with identical C shown in Fig. 8¹³SSNMR spectrum.

In one embodiment, the present invention relates to the polymorphic A of the toluene fulfonate of compound (1).This form is The nonsolvated forms of the toluene fulfonate of compound (1).In a specific embodiment, the toluene sulphur of compound (1) The feature of the form A of hydrochlorate be equivalent in X-ray powder diffraction figure case with spend measurement 7.2,9.3,13.7,14.3, 14.7, one or more peaks of 16.9,18.7,26.3 and 26.9 (± 0.2) 2- θ values.In another specific embodiment, The form A of the toluene fulfonate of compound (1), which is further characterized in that, to be equivalent in X-ray powder diffraction figure case to spend measurement 6.0,28.0 and 27.5 (± 0.2) 2- θ values one or more peaks.In another specific embodiment, compound (1) The form A of toluene fulfonate be characterized by XRPD pattern, have on the position listed in such as the following table 14 with 2- θ ± 0.2 characteristic peak indicated.In another specific embodiment, the feature of the form A of the toluene fulfonate of compound (1) is With XRPD pattern substantially identical with Fig. 9.XRPD pattern is obtained using Cu K alpha ray.

In another embodiment, the present invention relates to the HCl salt 1/2H of prepare compound (1)₂Form A, the chemical combination of O The HCl salt 3H of object (1)₂The form D of O, the form D of the HCl salt of compound (1), compound (1) form A and compound (1) Toluene fulfonate form A method.

The HCl salt 1/2H of compound (1)₂The form A of O can pass through mixing (such as stirring) hydrogen chloride (HCl) and change Close object (1) preparation.Compound (1) can be solvation, non-solvated, unbodied or crystallization.It can be by compound (1) solution, slurry or suspension mixes in the solvent system for including water and one or more organic solvents with HCl, wherein The solvent system, which has, is equal to or more than 0.05 and the water activity equal to or less than 0.85, i.e. 0.05-0.85.As this field Known the term as used herein " water activity " (a_w) refer to the measured value of the capability state of water in solvent system.It is defined as The vapour pressure of liquid divided by the pure water at identical temperature vapour pressure.Specifically, it is defined asWherein p is that water exists Vapour pressure in the substance, and p_oIt is the vapour pressure of pure water at the same temperature；It or is a_w=l_w×x_w, wherein l_wIt is the activity of water Coefficient, and x_oIt is molar fraction of the water in water section.For example, pure water has 1.0 water activity value.Water activity value can be typical Ground is obtained using electric-capacity moisture metre or cold-spot hygrometer.Different types of water activity measuring instrument is also commercially available.Alternatively, The water activity value of the mixture of two or more solvents can the known water activity value of solvent-based amount and solvent calculate.

The crystallization example of compound (1) includes the form A of compound (1).The example of the solvate of compound (1) includes The solvate of following solvent: 2-MeTHF, DMAC N,N' dimethyl acetamide, N,N-dimethylformamide, methanol, dimethylbenzene, third Ketone, 2- butanol, methyl acetate, 1- amylalcohol, 2- propyl alcohol, tetrahydrofuran, methyltetrahydrofuran, dimethyl acetamide, N, N- diformazan Base formamide, 1,4- dioxanes, 1- amylalcohol, 2- methyl-1-propyl alcohol, methyl ethyl ketone, 3- methyl-1-butanol, heptane, formic acid second Ester, n-butyl alcohol, acetic acid and ethylene glycol.In a specific embodiment, solvate (such as the chemical combination of 2-MeTHF is used Object (1) 1 (2-MeTHF)).

It is suitable for the HCl salt 1/2H of prepare compound (1)₂The solvent system of the form A of O can be by the water of various concentration It is combined into the group of organic solvent, wherein the water activity of the solvent system is equal to or more than 0.05 and equal to or less than 0.85 (0.05-0.85).In a specific embodiment, the value of water activity is 0.4-0.6.Suitable organic solvent includes the world Drug registration coordination committee guideline (International Conference on Harmonization Guidelines the II class or Group III organic solvent listed in).The specific example of suitable II class organic solvent include chlorobenzene, Hexamethylene, 1,2- dichloroethanes, methylene chloride, 1,2- dimethoxy-ethane, DMAC N,N' dimethyl acetamide, N, N- dimethyl formyl Amine, 1,4- dioxanes, cellosolvo, formamide, hexane, 2-methyl cellosolve, methyl butyl ketone, hexahydrotoluene, N- Methyl pyrrolidone, nitromethane, pyridine, sulfolane, tetrahydrofuran (THF), naphthane, toluene, 1,1,2- trichloro ethylene and Dimethylbenzene.The specific example of suitable Group III organic solvent includes: acetic acid, acetone, anisole, n-butyl alcohol, 2- butanol, acetic acid Butyl ester, t-butyl methyl ether, cumene, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3- methyl-1-butanol, methyl Ethyl ketone, methyl iso-butyl ketone (MIBK), 2- methyl-1-propyl alcohol, ethyl acetate, ether, Ethyl formate, pentane, 1- amylalcohol, 1- propyl alcohol, 2- propyl alcohol and propyl acetate.In a specific embodiment, the organic solvent of the solvent system is selected from: chlorobenzene, hexamethylene Alkane, 1,2- dichloroethanes, methylene chloride, 1,2- dimethoxy-ethane, hexane, 2-methyl cellosolve, methyl butyl ketone, methyl ring Hexane, nitromethane, naphthane, dimethylbenzene, toluene, 1,1,2- trichloroethanes, acetone, anisole, n-butyl alcohol, 2- butanol, second Acid butyl ester, t-butyl methyl ether, cumene, ethyl alcohol, ethyl acetate, ether, Ethyl formate, heptane, isobutyl acetate, isopropyl acetate Ester, methyl acetate, 3- methyl-1-butanol, methyl ethyl ketone, 2- methyl-1-propyl alcohol, pentane, 1- propyl alcohol, 1- amylalcohol, 2- propyl alcohol, Propyl acetate, tetrahydrofuran and methyltetrahydrofuran.In another specific embodiment, the solvent system is organic molten Agent is selected from cellosolvo, ethylene glycol, methanol, 2-methyl cellosolve, n-butyl alcohol, 2- butanol, 3- methyl-1-butanol, 2- first Base -1- propyl alcohol, ethyl alcohol, 1- amylalcohol, 1- propyl alcohol, 2- propyl alcohol, methyl butyl ketone, acetone, methyl ethyl ketone, methyl iso-butyl ketone (MIBK), Butyl acetate, isobutyl acetate, isopropyl acetate, methyl acetate, ethyl acetate, propyl acetate, pyridine, toluene and dimethylbenzene. In another embodiment, the organic solvent is selected from acetone, n-propanol, isopropanol, isobutyl acetate and acetic acid.Another In one embodiment, the organic solvent is selected from acetone and isopropanol.In another specific embodiment, the solvent System includes water and acetone.In another specific embodiment, the solvent system includes water and isopropanol.

The HCl salt 1/2H of prepare compound (1)₂The form A of O can be carried out at any suitable temperature.Typically, 5 DEG C -75 DEG C at a temperature of carry out.In a specific embodiment, carried out at a temperature of 15 DEG C -75 DEG C.At another In specific embodiment, carried out at a temperature of 15 DEG C -60 DEG C.In another specific embodiment, in 15 DEG C of -35 DEG C of temperature Degree is lower to carry out.In another specific embodiment, the preparation is at a temperature of 5 DEG C -75 DEG C with 0.4-0.6 water activity value Solvent system in carry out.In another specific embodiment, the preparation is at a temperature of 15 DEG C -75 DEG C with 0.4- It is carried out in the solvent system of 0.6 water activity value.In another specific embodiment, the preparation is in 15 DEG C of -60 DEG C of temperature Under carried out in the solvent system of the water activity value with 0.4-0.6.In another specific embodiment, the preparation is 15 It is carried out in the solvent system of the water activity value with 0.4-0.6 at a temperature of DEG C -35 DEG C.

Hydrogen chloride (HCl) can be imported as solution or gas.One example of suitable chlorination hydrogen source is comprising accounting for The aqueous solution of the hydrogen chloride of aqueous solution weight 30-40wt% (such as 34wt%-38wt%).

The HCl salt 3H of compound (1)₂The form D of O by include water solvent system or include water and one kind or it is more HCl and compound (1) preparation are mixed in the solvent system of kind organic solvent, are equal to or more than wherein the solvent system has The water activity of 0.9 (>=0.9).The mixture can be solution, slurry or suspension.Compound (1) can be solvation, non- It is solvation, unbodied or crystallization.Alternatively, can be by including the solvent system of water or including water and one or more The HCl salt 1/2H of compound (1) is stirred in the solvent system of organic solvent₂Prepared by the form A of O, wherein the solvent system With the water activity for being equal to or more than 0.9.Typically, pure water has 1.0 water activity value.Therefore, the water with 0.9-1.0 is living The solvent system of degree is suitable for the HCl salt 3H of prepare compound (1)₂The form D of O.In a specific embodiment, mix It closes or stirring carries out (18 DEG C -25 DEG C) at ambient temperature.In another specific embodiment, it mixes or stirs 15 It is carried out at a temperature of DEG C -30 DEG C.In another specific embodiment, it mixes or stirs in 20 DEG C -28 DEG C (such as 25 DEG C) temperature Degree is lower to carry out.It is used to form the HCl salt 3H of compound (1)₂The suitable organic solvent of the form D of O, including specific example is such as The above-mentioned HCl salt 1/2H to compound (1)₂Described in the form A of O.In another specific embodiment, the solvent system System includes water and acetone.In another specific embodiment, the solvent system includes water and isopropanol.

It can be by making the HCl salt 1/2H of compound (1)₂The HCl salt form of form A dehydration prepare compound (1) of O D.Dehydration can carry out in any suitable manner, such as heating or dry nitrogen purification or both.

The form A of compound (1) with prepare through the following steps: (a) stir amorphous compound (1) or compound (1) Mixture of the solvate (such as 2-MeTHF solvate of compound (1)) in the solvent system for including water and ethyl alcohol. The mixture can be solution or slurry.In a specific embodiment, whipping step 18 DEG C -90 DEG C at a temperature of into Row.In another specific embodiment, whipping step (a) carries out under the reflux temperature of solvent system.In another tool In the embodiment of body, the solvent system includes water 5-15wt%.The example of the solvate of compound (1) is as described above. In a specific embodiment, the solvate (such as compound (1) 1 (2-MeTHF)) of 2-MeTHF is used.

In another embodiment, the method for the form A of prepare compound (1) also includes: (b) is stirred in nitromethane The amorphous form of compound (1) is mixed, the crystal seed of the form A of compound (1) is formed；(c) by the form A's of compound (1) Crystal seed is added in mixture obtained in mixing step (a).In a specific embodiment, this method also includes: (b) The amorphous form that compound (1) is stirred in nitromethane, forms the crystal seed of the form A of compound (1)；(c) by mixing step Suddenly mixture obtained in (a) is cooled to the temperature of 18 DEG C -60 DEG C (such as 50-55 DEG C or 55 DEG C)；(d) by compound (1) Form A the mixing step (c) that is added to of crystal seed in.In another specific embodiment, this method also includes Water is added, then adds the crystal seed of the form A of compound (1) into the mixture obtained by reflow step, dosage makes The solvent system obtained after addition water includes the water of 15-25wt%.In another specific embodiment, this is non-also comprising inciting somebody to action Water is added in the mixture of the crystal seed of the form A including compound (1), and dosage makes the solvent system obtained after the addition of water System includes the water of 35-45wt%.In another specific embodiment, this method also include after the addition of water, will include change The mixture for closing the crystal seed of the form A of object (1) is cooled to 0 DEG C of -10 DEG C of temperature.

In a specific embodiment, the crystal seed of the form A of compound (1) can use the 2-MeTHF of compound (1) Solvate is prepared in nitromethane.It in one embodiment, include accounting for the solvent for the solvent system of reflow step The water of identical weight 5-15wt% (such as 8wt%, 10wt% or 12wt%).

The form A of the toluene fulfonate of compound (1) can pass through stirring amorphous compound (1) or compound (1) Solvate ((such as 2-MeTHF solvate of compound (1)), p- toluenesulfonic acid and the solvent system including acetonitrile it is mixed Close object preparation.In a specific embodiment, mixing or whipping step carry out at ambient temperature.It is specific at another In embodiment, mixing or whipping step 15-30 DEG C at a temperature of carry out.In another specific embodiment, it mixes Or whipping step 20-30 DEG C (such as 25 DEG C) at a temperature of carry out.The suitable example of the solvate of compound (1), packet Specific example is included as described in the above-mentioned form A prepare compound (1).

In another embodiment, the present invention relates to the 2-MeTHF solvates of compound (1).It is specific at one In embodiment, which includes the 2-MeTHF/ compound (1) of 0.5-1.5 equivalent, such as the 2-MeTHF/ization of 1 equivalent It closes object (1).In a specific embodiment, which includes the 2-MeTHF of 1 equivalent and is characterized by XRPD figure Case has the feature indicated in the position of following 8.4,9.7,16.7,16.9,17.4,21.0,22.3 and 25.7 with θ ± 0.2 2- Peak.In another specific embodiment, which includes the 2-MeTHF of 1 equivalent and is characterized by having some tables The peak XRPD listed in 12 is characterized by XRPD pattern as shown in Figure 10.

In another embodiment, the present invention covers the amorphous shape of compound (1) and its pharmaceutically acceptable salt Formula, such as the amorphous HCl salt and amorphous compound (1) of compound (1).In another embodiment, the present invention is also contained The form B of lid compound (1) hydrate.The form B of compound (1) hydrate and form A of compound (1) is the isomorphism, is shown Show the peak XRPD identical with the form A of compound (1), but formed in the absence of water, for example, with water activity be greater than 0.6, Such as in 0.6-1.0, system at ambient temperature.

The present invention covers the polymorphic of above compound (1), is for example changing for unpack format, pure form or with other materials The other forms (i.e. the amorphous form of compound (1), form A etc.) or arbitrary substance for closing object (I) are solid compositions when mixing The mixture of object.

In one aspect, the present invention provides polymorphic, such as the HCl salt 1/2H of compound (1)₂Form A, the chemical combination of O The 3H of the HCl salt of object (1)₂O form D, the form D of the HCl salt of compound (1), the form A of compound (1), compound (1) The form A of the toluene fulfonate of the form B and compound (1) of hydrate, for isolated solid form.On the other hand, originally Invention provides the amorphous form of compound (1) and its pharmaceutically acceptable salt, such as the amorphous HCl salt of compound (1) With amorphous compound (1), for isolated solid form.

On the other hand, the present invention provides polymorphic, such as the HCl salt 1/2H of compound (1)₂The form A of O, change Close the HCl salt 3H of object (1)₂The form D of O, the form D of the HCl salt of compound (1), compound (1) form A, compound (1) the form A of the toluene fulfonate of the form B of hydrate and compound (1) is pure form.This pure form refers to specifically Polymorphic account for 95% (w/w) or more, such as 98% (w/w) or more, 99% (w/w%) or more, 99.5% (w/w) or more or 99.9% (w/w) or more.On the other hand, provide compound (1) or its pharmaceutically acceptable salt it is amorphous shown in, be Pure form.This pure form refers to that amorphous form accounts for 95% (w/w) or more, such as 98% (w/w) or more, 99% (w/ W% more than), 99.5% (w/w) or more or 99.9% (w/w) or more.

More specifically, the present invention provide composition forms polymorphic or polymorphic and one or more other crystallizations, Solvate, amorphous or other polymorphous form of mixtures each or its are arbitrary combines.For example, implementing at one In scheme, the composition includes the HCl salt 1/2H of compound (1)₂The form A of O is another with one or more compounds (1) Outer polymorphic, such as the HCl salt of amorphous form, solvate, the form D of the HCl salt of compound (1), compound (1) 3H₂The form D of O, the form A of compound (1) and/or other forms or its arbitrary combination.Similarly, in another embodiment party In case, the composition includes the HCl salt 3H of compound (1)₂The form D of O and one or more compounds (1) it is other Polymorphic, such as the HCl salt 1/2H of amorphous form, solvate, compound (1)₂The HCl of the form A of O, compound (1) The form D of salt, the form A of compound (1) and/or other forms or combinations thereof.Similarly, in another embodiment, institute State the form D for the HCl salt that composition includes compound (1) and the other polymorphic of one or more compounds (1), such as nothing Amorphous form, solvate, compound (1) HCl salt 1/2H₂The HCl salt 3H of the form A of O, compound (1)₂The form of O F, the form A of compound (1) and/or other forms or combinations thereof.In another embodiment, the composition includes chemical combination The form A of object (1) and the other polymorphic of one or more compounds (1), such as amorphous form, hydrate, solvent close Object and/or other forms or combinations thereof.In another embodiment, the composition includes the toluenesulfonic acid of compound (1) The form A of salt and the other polymorphic of one or more compounds (1), such as amorphous form, hydrate, solvate And/or other forms or combinations thereof.More specifically, the composition may include trace to 100% specific polymorphic or Any amount, for example, 0.1%-0.5%, 0.1%-1%, 0.1%-2%, 0.1%-5%, 0.1%-10%, 0.1%-20%, The specific polymorphic of 0.1%-30%, 0.1%-40% or 0.1%-50% weight, the total amount with compound in composition (1) are Benchmark.Alternatively, the composition may include at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, The specific polymorphic of 99.5% or 99.9% weight, on the basis of the total amount of compound in composition (1).

For purposes of the present invention, chemistry is identified according to the periodic table of elements of the 75th edition " physical chemistry handbook " CAS version Element.In addition, at " Organic Chemistry ", Thomas Sorrell, University Science Books, Sausolito:1999 and " March ' s Advanced Organic Chemistry ", the 5th edition, editor: Smith, M.B. and The general provisions of organic chemistry are described in March, J., John Wiley&Sons, New York:2001, entire contents are led to accordingly It crosses and is incorporated by.

Unless otherwise indicated, the structure described herein is also intended to all isomeries including structure (for example, enantiomerism, non- Enantiomerism, cis- anti-, conformation and rotational isomeric) form.For example, R the and S configuration of each center of asymmetry, (Z) and (E) double bond Isomers and (Z) and (E) conformer are included in the present invention, except non-specifically drawing only one isomers.As this field Technical staff it will be appreciated that substituent group can be rotated freely around any rotatable key.For example, being depicted asSubstituent group also table Show

Therefore, the single three-dimensional chemical isomer of the compounds of this invention and optical siomerism, diastereoisomer, it is cis/trans, Conformation and rotational isomeric mixture are within the scope of the present invention.

Unless otherwise directed, otherwise all tautomeric forms of the compounds of this invention are within the scope of the present invention.

In addition, unless otherwise indicated, the structure described herein is also intended to including only there is one or more rich in same position Different compound in terms of the atom of element.For example, except with deuterium or tritium replacement hydrogen or with being rich in¹³C- or¹⁴C- carbon replacement carbon with Outside, there is the compound of existing structure within the scope of the present invention.This compound can be used as point in (for example) biological standardization Analysis tool or probe.This compound, especially deuterium (D) analog is also useful in the treatment.

The compound of the present invention is defined with its chemical structure and/or chemical name herein.When pass through chemical structure and chemistry Title refers to compound, and when chemical structure and chemical name conflict, the characteristic of compound is determined by chemical structure.

Those skilled in the art can be appreciated that the compound of the present invention may include a chiral centre.Thus lead to Formula compound can have (i.e. (+) or (-) enantiomer) with two different optical isomers.All such enantiomers and its mixed Closing object includes that racemic mixture is included in the scope of the invention.Method well-known in the art, such as hand can be passed through Property HPLC, enzyme split and chiral auxiliary obtain single optical isomer or enantiomer.

In one embodiment, the compound of the present invention of single enantiomer form is provided, at least 95%, at least 97% and at least 99% without corresponding enantiomer.

In another embodiment, the compound of the present invention is (+) enantiomeric form, and at least 95% without corresponding (-) enantiomer.

In another embodiment, the compound of the present invention is (+) enantiomeric form, and at least 97% without corresponding (-) enantiomer.

In another embodiment, the compound of the present invention is (+) enantiomeric form, and at least 99% without corresponding (-) enantiomer.

In another embodiment, the compound of the present invention is (-) enantiomeric form, and at least 95% without corresponding (+) enantiomer.

In another embodiment, the compound of the present invention is (-) enantiomeric form, and at least 97% without corresponding (+) enantiomer.

In another embodiment, the compound of the present invention is (-) enantiomeric form, and at least 99% without corresponding (+) enantiomer.

II. the purposes of compound (1) and its pharmaceutically acceptable salt

One aspect of the present invention relates generally to compound (1) and its pharmaceutically acceptable salt, including above-mentioned various solid Body form (such as the HCl salt 1/2H of compound (1)₂The HCl salt 3H of the form A of O, compound (1)₂The form D of O, chemical combination The toluene of the form D of the HCl salt of object (1), the form A of compound (1), the form B of compound (1) hydrate and compound (1) The form A of sulfonate) purposes, the purposes is for inhibiting the influenza virus in biological sample or patient to replicate, reduce biology The amount (reducing virus titer) of influenza virus and the influenza of patient is treated in sample or patient.Unless hereinafter separately referring specifically to Show, otherwise compound (1) and its pharmaceutically acceptable salt, including above-mentioned various solid forms (such as the HCl of compound (1) Salt 1/2H₂The HCl salt 3H of the form A of O, compound (1)₂Form D, the compound of the form D of O, the HCl salt of compound (1) (1) the form A of the toluene fulfonate of the form B and compound (1) of form A, compound (1) hydrate) it is commonly referred to as chemical combination Object.

In one embodiment, the present invention relates generally to (such as pharmaceutically acceptable group of compound disclosed herein Solvate form) purposes in any of the above-described specified application.

In yet another embodiment, compound disclosed herein can be used for reducing biological sample (for example, infected cell Culture) or mankind's (for example, virus titer of patient lungs) in virus titer.

Term " symptom that influenza virus connects mediation ", " influenza infection " or " influenza " as used herein be used interchangeably with Refer to the disease as caused by influenza virus infection.

Influenza is that the infectious disease of birds and mammal is influenced as caused by influenza virus.Influenza virus is positive myxovirus The RNA virus of section, Orthomyxoviridae family include 5 categories: influenza A, Type B influenza virus, c-type influenza virus, ISA virus With the high soil virus of support.Influenza A category has a kind, influenza A, can be based on antibody responses to these viruses by A type Influenza virus is subdivided into different serotypes H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3 and H10N7.A Another example of type influenza virus includes H3N8 and H7N9.Type B Influenza Virus has a kind, Type B influenza virus.Type B influenza Almost specially infection the mankind and compared to A type influenza it is less common.C-type Influenza Virus has a kind, and c-type influenza virus infects The mankind and pig and can cause serious disease and endemic conditions disease.However, c-type influenza compared to other types it is less common and Usually seem the Milder disease for causing children.

In some embodiments of the present invention, influenza or influenza virus are related to A or Type B influenza virus.In the present invention Some embodiments in, influenza or influenza virus are related to influenza A.In some specific embodiments of the invention In, influenza A H1N1, H2N2, H3N2 or H5N1.In some embodiments of the present invention, influenza A is H1N1, H3N2, H3N8, H5N1 and H7N9.In some specific embodiments of the invention, influenza A H1N1, H3N2, H3N8 and H5N1.

In the mankind, the common sympton of influenza is shiver with cold, fever, pharyngitis, courbature, severe headache, cough, weakness and complete Body is uncomfortable.In the case where even more serious, influenza causes pneumonia that can be fatal, especially in child and the elderly.Although often Often obscure with common cold, but influenza is even more serious disease and caused by different types of virus.Especially exist In children, influenza can cause nausea and vomiting, but these symptoms are more characterized in that unrelated enterogastritis, sometimes referred to as " stomach flu " or " 24 hours influenzas ".

1-2 days after infection, flu symptom very can suddenly start.Usual onset symptoms are shiver with cold or chilly, but are infected Middle fever is also common early symptom, and body temperature is 38-39 DEG C (about 100-103 °F).Many people are in a bad way to crouching Bed a couple of days, overall pain, back and leg are more serious.Flu symptom may include: physical distress, especially joint and throat, Extreme cold and fever, fatigue, headache, irritation shed tears, eyes, skin (especially face), mouth, throat and nose it is rubescent, Abdominal pain (in the children for suffering from Type B influenza).Flu symptom and many pathogen (" parainfluenza ") are non-specific concurrent.In general, needing Laboratory data is wanted to make a definite diagnosis.

Medical treatment or pathology shape to refer to influenza virus mediation is used interchangeably in term " disease ", " illness " and " symptom " herein Condition.

Term " subject " as used herein and " patient " convertibly use.Term " subject " and " patient " refer to Object (for example, the birds such as chicken, quail or turkey or mammal), " mammal " especially including non-primate (for example, ox, pig, horse, sheep, rabbit, cavy, rat, cat, dog and mouse) and primate are (for example, monkey, chimpanzee and people Class), the more particularly mankind.In one embodiment, subject is non-human animal, such as domestic animal (for example, horse, ox, pig Or sheep) or pet (for example, dog, cat, cavy or rabbit).In a preferred embodiment, subject is " mankind ".

Term " biological sample " as used herein includes but is not limited to cell culture or its extract, from mammal The biopsy substance or its extract of acquisition, blood, saliva, urine, excrement, sperm, tears or other body fluid or its extraction Object.

Term " infection multiplicity " or " MOI " as used herein are infectious agent (for example, bacteriophage or virus) and infection pair As the ratio between (for example, cell).For example, infection multiplicity or MOI are when being related to a group and being vaccinated with the cell of infectious viral particle As the infectious viral particle quantity that is precipitated in a hole divided by target cell numbers present in the hole ratio of determination.

Term " duplication for inhibiting influenza virus " as used herein includes reducing the amount of virus replication (for example, reducing extremely Lack 10%) and prevent completely virus replication (that is, 100% reduces amount of virus replication).In some embodiments, influenza virus Duplication is suppressed at least 50%, at least 65%, at least 75%, at least 85%, at least 90% or at least 95%.

Influenza virus duplication can be measured by any appropriate methodology known in the art.For example, can measure in biological sample Influenza virus titer in (such as infection cell culture) or mankind's (for example, lung virus titer of patient's body).It is more special Not, for the detection based on cell, cell is cultivated in vitro in all cases, in test reagent existence or non-existence It is lower that virus is added in culture, and in due course between after assess viral dependent form terminal.For typical case's detection, it can make With the influenza strain A/Puerto Rico/8/34 that madin-Darby canine kidney cell (MDCK) and normal structure culture are suitable.At this Workable first kind cell detection depends on the death of target cell infection in invention, this is one and is known as cytopathic effect (CPE) process, wherein virus infection causes cellular resources to exhaust finally to dissolve with cell.In first kind cell detection, sense The sub-fraction cell (usual 1/10 to 1/1000) in micro titer plate well is contaminated, carries out virus at 48-72 hours multiple several times Then system uses the reduction measurement cell death quantity of the cell ATP compared with being uninfected by control.It is adoptable in the present invention Second class cell detection depends on proliferation of the virus-specific RNA molecule in infection cell, uses branch DNA hybridization method (bDNA) rna content is directly measured.In the second class cell detection, a small amount of cell is initially infected in the hole of microtiter plate, So that virus is replicated in infection cell and is traveled to other cell, then dissolve cell and measures viral RNA content.Usually exist Stop this detection after 18-36 hours early, and all target cells still survive.By with the spy on the hole for being fixed on detection plate Specific oligonucleotide probe hybridization, then amplified signal is viral to quantify and the other probe hybridization connecting with reporter enzyme RNA。

" virus titer " is the measurement of virus concentration as used herein.Serial dilution can be used with from essence in titre test On be only evaluated as positive or negative analysis method and obtain almost quantitative information.Titre with still generate the highest dilution gfactor phase just read It is corresponding；For example, first 8 times it is continuous twice dilution in just reading be converted into 1: 256 titre.Specific example is virus titer.For It determines titre, prepares several dilutions, such as 10^-1、10^-2、10^-3、10^-4,10^-5、10^-6、10^-7、10^-8Deng.Still infection cell Minimum virus concentration is virus titer.

Term " treatment " as used herein refers to therapeutic and prophylactic treatment.For example, therapeutic treatment includes due to applying Mitigated with one or more therapies (for example, one or more therapeutic agents (such as the compound of the present invention and composition)) or is improved Progress, severity and/or the duration for the symptom that influenza virus mediates, or improve the one kind for the symptom that influenza virus mediates Or a variety of symptoms (particularly, one or more distinguishable symptoms).In specific embodiments, therapeutic treatment includes improving influenza At least one of virus-mediated symptom can measure physical parameter.In other embodiments, therapeutic treatment includes passing through (example As) stablize distinguishable symptom and physiologically or both influenza virus is being inhibited to mediate physically or by (for example) stable physical parameter Symptom progress.In other embodiments, therapeutic treatment includes the infection for mitigating or stablizing influenza virus and mediate.It can be Using antiviral drugs to treat the people for having suffered from influenza to reduce the severity of symptom and to reduce them sick in community Number of days.

Term " chemotherapy ", which refers to, treats conditions or diseases using drug, such as small-molecule drug (rather than " vaccine ").

Term " prevention ", " preventive use " and " prophylactic treatment " feeling the pulse with the finger-tip as used herein is to prevent, without It is treatment or any medical treatment or public health program for curing disease.Term " preventing " as used herein refers to that reduction is obtained or sent out Open up the risk of specified symptom, or reduce or inhibit recurrence without disease but or may close illness people subject it is described Symptom.Term " chemoprophylaxis ", which refers to, prevents conditions or diseases using drug, such as small-molecule drug (rather than " vaccine ").

Preventive use as used herein includes for having detected that in the case of outburst to prevent in many in tight The place of people's close contact of weight influenza complications high risk is (for example, in hospital ward, day-care center, prison, sanatorium Deng) infect or propagate.It further include that influenza is being needed to protect but do not protected after vaccine inoculation (for example, due to siberian crabapple Unite weak) group in or when vaccine is unavailable for them, or when they due to side effect is unable to vaccine inoculation when use. It further include being used in 2 weeks after vaccine inoculation, because vaccine is still invalid during this period.Preventive use is also possible that treatment not Suffer from influenza or is not regarded as the people in complication high risk to reduce infection influenza and be transmitted to influenza and his close contact High-risk people (for example, medical staff, sanatorium staff etc.) chance.

According to US CDC, influenza " outburst " is defined as in a group people close to each other (such as set supporting sexual life In the same area applied, it is medium in the same family) in 48-72 hours acute febrile respiratory disease (AFRI) increase suddenly it is super Cross normal background rate or when subject's test any in the group analyzed is that influenza is positive.It will be determined by any test method Flu casess be considered as outburst.

" cluster " is defined as in a group people close to each other (for example, in the same area, same for supporting sexual life facility In family etc.) interior 3 or more the AFRI diagnostic cost groups occurred at 48-72 hours.

" indicator case " used herein, " primary case " or " No. zero patient (patient zero) " are epidemiology tune The initial patient in population sampling looked into.For when referring to this patient, term to be not capitalized usually in epidemiological survey.When When about being used to refer to specific people rather than the name of that people for term in the report of particular survey, term is written as greatly No. zero trouble Person (Patient Zero).Scientist finds indicator case usually to determine how disease is propagated and burst period which kind of in spite of illness Source can control disease.Notice that indicator case is that instruction breaks out existing first patient.It can find case earlier, and labeled as the One, second, third etc..

In one embodiment, method of the invention is that especially have as caused by influenza infection simultaneously to patient Send out preventative or " preferential " measure of the people of the procatarxis of disease.Such as this paper term used in preferential use, " preferentially " etc. " preferential " preventive use in the case of having confirmed that " indicator case " or " outburst ", to prevent infection in community or group Rest part in propagate.

In another embodiment, method of the invention can be used as the member to community or group, the especially mankind " preferential " measure, to prevent transmission of infection.

" effective quantity " has guided the amount of expected biological respinse as used herein.In the present invention, it is contemplated that biological respinse is Inhibit influenza virus duplication, reduce influenza virus amount mitigation or improve the severity of influenza infection, the duration, Progress or breaking-out, prevent influenza infection from spreading, prevent influenza infection it is related indication recurrence, develop, breaking-out or into Exhibition, or enhancing or the prevention or therapeutic effect that improve another anti influenza therapy of infection used.The chemical combination applied to subject The exact amount of object will depend on administration mode, the feature of the type of infection and severity and subject, such as health status, year Age, gender, weight and the tolerance to drug.Technical staff will determine suitable dosage according to these and other factor.When With other antivirotics be administered in combination when, such as with Tamiflu be administered in combination when, " effective quantity " of second of reagent will take Certainly in the type of drug used.The suitable dosage of known approved reagent and technical staff can according to the symptom of subject, control The amount of the type and the compound described herein used for the treatment of symptom is adjusted.In the case where the amount of pointing out is not known, should take Effective quantity.For example, describedization disclosed herein can be applied to subject by the dosage range of about 0.01-100mg/kg body weight/day It closes object and does therapeutic or prophylactic treatment.

In general, dosage can be selected according to Multiple factors, and the severity including treating illness and illness, the spy of use Determine the activity of compound, the particular composition of use, age, weight, health status, gender and the diet of patient, administration time, The excretion rate of administration method and the specific compound of use, the hepatic and renal function of subject, the special compound of use or its salt, are controlled The duration is treated, the drug used in conjunction with the specific compound of use or simultaneously and medically well-known similar factor. Technical staff may easily be determined and designated treatment, prevent, inhibit (complete or partial) or prevent progression of disease needed for this paper institute State the effective quantity of compound.

The dosage range of compound described herein be about 0.01-100mg/kg body weight/day, 0.01-50mg/kg body weight/day, 0.1-50mg/kg body weight/day or 1-25mg/kg body weight/day.It should be understood that daily total amount can be applied or can be pressed by single dose Multidose application, for example, 2 times a day (for example, every 12 hours), 3 times a day (for example, for every eight hours) or 4 times a day (for example, Every 6 hours).

In some embodiments, compound as described herein (such as compound (1) and its pharmaceutically acceptable salt, Including various solid forms (such as the HCl salt 1/2H of compound (1)₂The HCl salt 3H of the form A of O, compound (1)₂O's Form D, the form D of the HCl salt of compound (1), the form A of compound (1), compound (1) hydrate form B and compound (1) the form A of toluene fulfonate)) dosage be 100mg-1,600mg, such as 400mg-1,600mg or 400mg-1, 200mg.By every kind of dosage daily 1 time (QD), such as 2 times (such as every 12 hours (BID)) or (such as q8h 3 times a day (TID)).Note that as needed, any combination of QD, BID and TID can be used, for example, on day 1 when BID, subsequent QD.

In some embodiments, compound as described herein (such as compound (1) and its pharmaceutically acceptable salt, Including various solid forms (such as the HCl salt 1/2H of compound (1)₂The HCl salt 3H of the form A of O, compound (1)₂O's Form D, compound (1) HCl salt form D) dosage be 100mg-1,600mg, such as 400mg-1,600mg or 400mg- 1,200mg.By every kind of dosage daily 1 time (QD), for example, 2 times (such as every 12 hours (BID)) or 3 times a day (such as q8h(TID)).Note that as needed, any combination of QD, BID and TID can be used, for example, on day 1 when BID, then QD；Or loading dose is used when on day 1, BID at the 2nd day, then QD.

In a specific embodiment, the dosage of compound described herein is 400mg-1,600mg, 400mg-1, 200mg or 600mg-1,200mg, one time a day.In another specific embodiment, the dosage of compound described herein is 400mg-1,600mg, 400mg-1,200mg or 300mg-900mg, 2 times a day.In another specific embodiment, originally The dosage of the text compound is 400mg-1,000mg, one time a day.In another specific embodiment, described hereinization Close the dosage of object for 600mg-1,000mg, one time a day.In another specific embodiment, the agent of compound described herein Amount is 600mg-800mg, one time a day.In another specific embodiment, the dosage of compound described herein is 400mg- 800mg, 2 times a day (such as every 12 hours 400mg-800mg).In another specific embodiment, described hereinization The dosage for closing object is 400mg-600mg, 2 times a day.

In some embodiments, using loading dose scheme.In a specific embodiment, treat on day 1 When, use 400mg-1, the loading dose of 600mg.In another specific embodiment, it when treating on day 1, uses The loading dose of 600mg-1,600mg.In another specific embodiment, when treating on day 1, using 800mg-1, The loading dose of 600mg.In another specific embodiment, when treating on day 1, using 900mg-1,600mg's is negative Lotus dosage.In another specific embodiment, when treating on day 1,900mg-1, the loading dose of 200mg are used.? In another specific embodiment, when treating on day 1, the loading dose of 900mg is used.In another specific embodiment party In case, when treating on day 1, the loading dose of 1,000mg is used.In another specific embodiment, it treats on day 1 When, use the loading dose of 1,200mg.

In a specific embodiment, the dosage of compound as described herein on day 1 when use 600mg- The loading dose of 1,600mg and the regular dosage that 300mg-1,200mg is used during remaining treatment.Every kind can be taken Regular dosage, one time a day, 2 times a day or 3 times a day or it arbitrary is combined.In another specific embodiment, it uses The loading dose of 900mg-1,600mg such as 900mg, 1,200mg or 1,600mg.In another specific embodiment, make With the loading dose of 900mg-1,200mg, such as 900mg or 1,200mg.In another specific embodiment, 400mg- The regular dosage of 1,200mg such as 400mg, 600mg or 800mg are for remaining treatment time limit.In another specific implementation In scheme, the regular dosage of 400mg-1,000mg are for remaining treatment time limit.In another specific embodiment, The regular dosage of 400mg-800mg is for remaining treatment time limit.In another specific embodiment, using 300mg- The regular dosage of 900mg, 2 times a day.In another specific embodiment, using 600mg-1, the regular dosage of 200mg, One time a day.When in another specific embodiment, on day 2, using the regular dosage of 600mg, 2 times a day, followed by 600mg, one time a day, for remaining treatment time limit.

For therapeutic treatment, for example, can paresthesia epilepsy (for example, nasal obstruction, sore-throat, cough, pain, fatigue, Headache and shiver with cold/perspiration) it is applied in (or in 40 hours or less than 2 days or less than 1.5 days or 24 hours) 48 hours to patient Compound described herein.Alternatively, for therapeutic treatment, for example, can be applied in paresthesia epilepsy 96 hours to patient Compound as described herein.Therapeutic treatment sustainable any suitable time, such as 3 days, 4 days, 5 days, 7 days, 10 days, 14 days Deng.For the prophylactic treatment of community's burst period, such as this can be applied to patient in indicator case's paresthesia epilepsy 2 days The text compound, and any suitable time can be continued, such as 7 days, 10 days, 14 days, 20 days, 28 days, 35 days, 42 days etc., Until entire Influenza flu season.Influenza flu season is the time limit recurred every year, it is characterised in that influenza outbreak.Influenza activity Sometimes it is expected that and even can geographically track.When influenza activity main in each season starts because of geographical location Difference and when changing, in any specific place, these less disease popularities, which generally take, reaches peak value and again for 3-4 weeks By 3-4 weeks obvious decrease.Typically, at the Center for Disease Control (Centers forDisease Control) (CDC) It acquires, collect and analyzes the information in relation to influenza activity year and be produced from the report weekly of October to mid-April.

In one embodiment, therapeutic treatment continues 1 day to entire Influenza flu season.In a specific embodiment In, therapeutic treatment continues -14 days 3 days.In another specific embodiment, therapeutic treatment continues -14 days 5 days.? In another specific embodiment, therapeutic treatment continues -10 days 3 days.In another specific embodiment, therapeutic Treatment continues -10 days 4 days.In another specific embodiment, therapeutic treatment continues -10 days 5 days.It is specific at another Embodiment in, therapeutic treatment continues -7 days 4 days (such as 4 days, 5 days, 6 days or 7 days).In another specific embodiment party In case, therapeutic treatment continues -7 days 5 days (such as 5 days, 6 days or 7 days).It is preventative to control in a specific embodiment Treatment continues to entire Influenza flu season.

In a specific embodiment, on day 1 when, by compound as described herein use 900mg-1,600mg Loading dose be applied to patient -14 days 3 days (such as -14 days 5 days), and use 300mg-1, the regular dosage of 200mg is used The time limit is treated in remaining.When in another specific embodiment, on day 1, compound as described herein is used The loading dose of 900mg-1,200mg are applied to patient -14 days 3 days (such as -14 days 5 days), and use 400mg-1,000mg Regular dosage for remaining treatment time limit.It, will be as described herein when in another specific embodiment, on day 1 Compound uses 900mg-1, and the loading dose of 200mg is applied to patient -14 days 3 days (such as -14 days 5 days), and uses The regular dosage of 400mg-800mg is for remaining treatment time limit.It, will when in another specific embodiment, on day 1 Compound as described herein uses 900mg-1, and the loading dose of 200mg is applied to patient -14 days 3 days (such as -14 days 5 days), And using the regular dosage of 400mg-800mg for remaining treatment time limit.Every kind of dosage can be taken, one time a day, daily 2 times or 3 times a day or it arbitrary is combined.

In a specific embodiment, on day 1 when, by compound as described herein use 900mg-1,600mg Loading dose be applied to patient -14 days 3 days, and use 600mg-1,000mg, regular dosage one time a day are used for remaining The treatment time limit.When in another specific embodiment, on day 1, compound as described herein is used into 900mg-1, The loading dose of 200mg is applied to patient -14 days 3 days, and use 600mg-800mg (such as 600mg, 650mg, 700mg, 750mg or 800mg), regular dosage one time a day is for remaining treatment time limit.In some embodiments, the sessions lasts - 10 days 4 days, -10 days 5 days or -7 days 5 days.

In a specific embodiment, on day 1 when, by compound as described herein use 900mg-1,600mg Loading dose be applied to patient -14 days 3 days, and be used for remaining using 400mg-800mg, regular dosage 2 times a day Treat the time limit.When in another specific embodiment, on day 1, compound as described herein is used into 900mg-1, The loading dose of 200mg is applied to patient -14 days 3 days, and using 400mg-600mg (such as 400mg, 450mg, 500mg, 550mg or 600mg), regular dosage 2 times a day is for remaining treatment time limit.In some embodiments, the sessions lasts - 10 days 4 days, -10 days 5 days or -7 days 5 days.

In a specific embodiment, on day 1 when, by compound as described herein use 900mg-1,200mg The loading dose of (such as 900mg or 1,200mg) is applied to patient 4 days or 5 days, and using 400mg-600mg (such as 400mg or 600mg), regular dosage 2 times a day is for remaining (such as-the 4 day the 2nd day or the 2nd day-the 5 in treatment time limit It).When in another specific embodiment, on day 1, compound as described herein is used into 900mg-1,200mg (example Such as 900mg or 1,200mg) loading dose be applied to patient 4 days or 5 days, and using 600mg-800mg (such as 600mg or 800mg), regular dosage one time a day is for remaining treatment time limit.

Various types of method of administration can be used in the present invention, and under the part of following entitled " method of administration " Detailed description.

III. conjoint therapy

It can be in the independent sheet using individually or in conjunction with the therapeutic agent such as antivirotic or vaccine suitable with another kind Compound (the method for the present invention or drug including its pharmaceutically acceptable salt or solvate (for example, hydrate) of invention Reach effective quantity in composition.When using " conjoint therapy ", the compounds of this invention and second amount of the first amount are used In addition suitable therapeutic agent (for example, antivirotic or vaccine) can reach effective quantity.

In another embodiment of the present invention, by the compound of the present invention and other therapeutic agent respectively with effective quantity (that is, If be administered alone, respectively to treat upper effective amount) application.In another embodiment, by the compound of the present invention and separately Outer therapeutic agent is respectively will not individually provide amount (asian treatment dosage) application of curative effect.It in yet another embodiment, can be by effective Amount application the compound of the present invention, and other therapeutic agent is applied by asian treatment dosage.In still another embodiment, it can be controlled by Asia It treats dosage and applies the compound of the present invention, and apply other therapeutic agent by effective quantity, such as suitable cancer therapeutic agent.

Term " in conjunction with " as used herein or " combined administration " are used interchangeably to refer to the therapy (example for using more than one Such as, one or more preventions and/or therapeutic agent).The use of term be not intended to limit to subject apply therapy (for example, prevention and/ Or therapeutic agent) sequence.

The compound for covering the first and second of amount that combined administration is applied in the way of substantially simultaneously is administered in combination, Such as it is applied by single drug composition, such as capsule or tablet that the first and second of amount ratio are fixed, or press multiple lists Only capsule or tablet application.In addition, this combined administration also covers and successively uses every kind of compound by any order.

In one embodiment, the present invention relates to use compound as described herein to inhibit to flow in biological sample or patient Influenza Virus duplication or treatment or the conjoint therapy for preventing patient's influenza infection.Therefore, pharmaceutical composition of the invention is also wrapped Including those includes the influenza virus inhibition of DNA replication of the invention in conjunction with the antiviral compound for showing anti-influenza virus activity Agent.

The application method of compound as described herein and composition of the invention further includes combining chemotherapy and the present invention Compound or composition, or combine the combination and another antivirotic and inoculation influenza epidemic disease of the compound of the present invention or composition Seedling.

When combined administration includes the compounds of this invention that the first amount is administered alone and the other therapeutic agent of second of amount When, it is being enough to apply compound close to the time for having expected effect.For example, when can produce the interval of the application of expected effect every time Between range can be a few minutes to a few hours, and the property of every kind of compound can be considered, such as effect, dissolubility, biological utilisation Rate, plasma half-life and dynamic characteristic.For example, can be spaced apart from each other in 24 hours, be spaced apart from each other 16 hours in any order It is interior, be spaced apart from each other in 8 hours, be spaced apart from each other in 4 hours, be spaced apart from each other in 1 hour or be spaced apart from each other the application present invention in 30min Compound and second of therapeutic agent.

More specifically, can to subject apply second therapy (such as prevention or therapeutic agent, such as anticancer agent) it Before (such as first 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 Hour, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks), concurrently or after which (such as 5 minutes later, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks) apply the first therapy (such as prevention or treatment Agent, such as the compound of the present invention).

It should be appreciated that the other therapeutic agent of the compounds of this invention and second of dosage of the first dosage of co-administration Method can produce enhancing or synergistic therapeutic action, and wherein synergy is greater than the present inventionization because of the first dosage of separate administration Close the accumulative action of the other therapeutic agent of object and second of dosage.

Term " collaboration " as used herein refers to the group of the compounds of this invention and another therapy (for example, prevention or therapeutic agent) It closes, this is more more effective than the additive effect of therapy.The synergistic effect of the combination (for example, prevention or combination of therapeutic agent) of therapy can permit Perhaps the therapy using one or more therapies of lower dosage and/or more low frequency is applied to subject.It can be using more It applies the therapy to the therapy (for example, prevention or therapeutic agent) of low dosage and/or more low frequency and can reduce and applied to subject The relevant toxicity of the therapy, without reducing the effect of the therapy in illness prevention, management or treatment.In addition, collaboration effect The effect of reagent in illness prevention, management or treatment should can be caused to improve.Finally, the combination of therapy is (for example, prevention or treatment The combination of agent) synergistic effect can avoid or reduce nocuousness relevant with any therapy is used alone or adverse side effect.

When the conjoint therapy carried out using the compounds of this invention of the invention is in conjunction with influenza vaccines, two kinds can be applied and controlled Agent is treated so that the interval time applied every time can be longer (for example, a couple of days, several weeks or several months).

The appropriate methodology of assessment drug interaction can be used to determine the presence of synergistic effect.Appropriate methodology includes (for example) Sigmoid-Emax formula (Holford, N.H.G. and Scheiner, L.B., Clin.Pharmacokinet.6:429-453 (1981)), Loewe be added formula (Loewe, S. and Muischnek, H., Arch.Exp.Pathol Pharmacol.114: 313-326 (1926)) He Zhongxiao formula (Chou, T.C.and Talalay, P., Adv.Enzyme Regul.22:27-55 (1984)).Above-mentioned each formula can application be together with experimental data to generate corresponding figure, to help to assess drug Combined effect.Above-mentioned corresponding figure relevant with formula is respectively concentration effect curve, equivalent line chart curve and group Hop index curve.

The specific example that can be administered in combination with compound described herein includes neuraminidase inhibitor (for example, Ao Sita WeiAnd zanamivir), viral ion channel (M2 albumen) retarding agent is (for example, Buddha's warrior attendant AmineAnd RimantadineWith antiviral agent described in WO 2003/015798 Object, including Japanese Toyama Chemical research and development T-705 (referring also to Ruruta etc., Antiviral Reasearch, 82: 95-102 (2009), " T-705 (flavipiravir) and related compounds:Novel broad-spectrum inhibitors of RNA viral infections.").In some embodiments, compound described herein can be with routine Influenza vaccines are administered in combination together.

In some embodiments, can by compound as described herein (such as compound (1) and its pharmaceutically can and Ei The salt killed, such as the HCl salt 1/2H of compound (1)₂The HCl salt 3H of the form A of O, compound (1)₂The form D of O, chemical combination The toluene of the form D of the HCl salt of object (1), the form A of compound (1), the form B of compound (1) hydrate and compound (1) The form A of sulfonate) it is co-administered with zanamivir.It in some embodiments, can be by compound as described herein and method Wella Wei (T-705) is co-administered.In some embodiments, compound as described herein can be applied jointly with Oseltamivir With.In some embodiments, compound as described herein and the different amine of amantadine or Buddha's warrior attendant can be co-administered.It can be by The dosage side's amine specified in sighting target label applies Oseltamivir.In some specific embodiments, 75mg can be applied, often Day 2 times；Or 150mg, one time a day.

Pharmaceutical composition

Compound described herein can be formulated as further including pharmaceutically acceptable carrier, diluent, auxiliary material or tax The pharmaceutical composition of shape agent.In one embodiment, the present invention relates to compounds and pharmacy comprising present invention described above The pharmaceutical composition of upper acceptable carrier, diluent, auxiliary material or excipient.In one embodiment, the present invention be comprising A effective amount of the compound of the present invention or its pharmaceutically acceptable salt and pharmaceutically acceptable carrier, diluent, auxiliary material or The pharmaceutical composition of excipient.Pharmaceutically acceptable carrier includes, for example, pharmaceutical diluents, excipient or just expected application What form suitably selected meets the carrier for facilitating medicinal practice.

" effective quantity " includes " therapeutically effective amount " and " prevention effective dose ".Term " therapeutically effective amount " refers to treatment and/or changes The influenza infection of the sentimental patient for having contaminated influenza is effectively measured.Term " prevention effective dose ", which refers to, to be prevented and/or generally subtracts The chance or range of few influenza infection outburst are effectively measured.In being described using part for the above entitled open compound A effective amount of specific example.

Pharmaceutically acceptable carrier may containing will not extra-inhibitory compound bioactivity inert fraction.Pharmacy Upper acceptable carrier answers bio-compatible, such as nontoxic, non-inflammatory, non-immunogenic or is once administered to patient without other bad Reaction or side effect.Standard pharmaceutical techniques can be used.

Pharmaceutically acceptable carrier, auxiliary material or excipient as used herein include any all suitable for required special agents Solvent, diluent or the other liquid excipients of type, dispersion or suspension adjuvant, surfactant, isotonic agent, thickener or cream Agent, preservative, solid binder, lubricant etc..The Pharmaceutical Sciences of Remington the 16th edition, E.W.Martin (Mack Publishing Co., Easton, Pa., 1980), which is disclosed, prepares pharmaceutically acceptable composition Used in various carriers and its known technology of preparing.In addition within the scope of any conventional carrier, medium and compound described herein It is incompatible, such as by generating any non-desired biological effect or with any of harmful way and pharmaceutically acceptable salt Other component interactions, purposes are taken into account within the scope of the present invention.Phrase " side effect " as used herein is covered The harmful detrimental effect of therapy (for example, prevention or therapeutic agent).Side effect is always undesirable, but it is undesirable not must It is harmful.The detrimental effect of therapy (for example, prevention or therapeutic agent) may be harmful uncomfortable or dangerous.Side effect includes but is not limited to Fever, shiver with cold, drowsiness, gastrointestinal toxicity (including stomach and enterelcosis and erosion), Nausea and vomiting, neurotoxicity, Poisoning kidney damage Evil, renal toxicity (including such as symptom such as necrosis of renal papillae and arteriosclerotic kidneys), hepatotoxicity wind agitation (including serum liver enzyme levels liter It is high), myelotoxicity (including oligoleukocythemia, bone marrow suppression, decrease of platelet and anaemia), dry, metallic taste, extend it is pregnant, Weakness, somnolence, pain (including courbature, ostalgia and headache), alopecia, inability, dizziness, extrapyramidal symptom, stationary seat cannot, the heart Dirty vascular disorder and sex dysfunction.

The some examples that can be used as the substance of pharmaceutically acceptable carrier include but is not limited to ion-exchanger, oxidation Aluminium, aluminum stearate, lecithin, haemocyanin (such as human serum albumins), buffer substance are (such as twin 80, phosphate, sweet Propylhomoserin, sorbic acid or potassium sorbate), the partial glyceride mixture of saturated vegetable fatty acid, water, salt or electrolyte (such as sulfuric acid Protamine, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride or zinc salt), silica gel, magnesium trisilicate, polyvinylpyrrolidone, polypropylene Acid esters, wax, polyethylene-polypropylene oxide-block copolymer, methylcellulose, hydroxypropyl methyl cellulose, lanolin, carbohydrate (such as lactose, dextrose and saccharose), starch (such as cornstarch and potato starch), cellulose and its derivates (such as Sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate), powdered tragacanth, malt, gel, talcum, excipient (such as Cupu oil and suppository wax), oily (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil), Ethylene glycol (such as propylene glycol or polyethylene glycol), ester (such as ethyl oleate and ethyl laurate), agar, buffer (such as hydrogen Magnesia and aluminium hydroxide), alginic acid, apirogen water, isotonic saline solution, Ringer's solution (Ringer's solution), ethyl alcohol With phosphate buffer and other non-toxic compatible lubricants (such as NaLS and magnesium stearate) and according to matching Judgement colorant, antitack agent, coating agent, sweetener and the fumet of people processed, preservative and antioxidant also are present in combination In object.

IV. method of administration

Can according to by control infection severity is oral, rectum, parenteral, in brain pond, in intravaginal, peritonaeum, part (such as With pass through pulvis, ointment or drops), oral cavity as mouth or nasal spray etc. to people or other animals application the above compound and Pharmaceutically acceptable composition.

For oral liquid dosage form include but is not limited to pharmaceutically acceptable emulsion, microemulsion, solution, suspending agent, Syrup and elixir.In addition to the active compound, liquid dosage form may contain inert diluent commonly used in the art, such as water or other Solvent, solubilizer and emulsifier, such as ethyl alcohol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, Ergol, the third two Alcohol, 1,3-BDO, dimethylformamide, oil (especially cottonseed oil, peanut oil, corn oil, embryo oil, olive oil, castor-oil plant Oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan aliphatic ester and its mixture.Except inert diluent Outside, Orally administered composition may also comprise auxiliary material, such as wetting agent, emulsification and suspending agent, sweetener, flavoring agent and fumet.

Injectable formulation can be prepared using suitable dispersion or wetting agent and suspending agent according to known technology, for example, it is sterile can Injection water or oil-suspending agent.Sterile injectable preparation is also likely to be the nothing in the acceptable diluent of nontoxic parenteral or solvent Solution in bacterium Injectable solution, suspending agent or emulsion, such as 1,3-BDO.It, can in acceptable excipient and solvent Using water, Ringer's solution, U.S.P. and isotonic sodium chlorrde solution.In addition, using sterile non-volatile oil by convention As solvent or suspension media.For this purpose, any tasteless fixed oil can be used, monoglycceride including synthesis or Diglyceride.In addition, fatty acid, such as oleic acid, it is used to prepare injection.

For example, can be filtered by bacteria-retaining filter or by being added in aseptic solid composite form, before use The fungicide in sterile water or other sterile injectable mediums is dissolved in or is scattered in as injectable formulation sterilizing.

For the effect for extending compound described herein, it is often desirable to slow down compound by the absorption subcutaneously or intramuscularly injected. This can be realized by using the crystal of poorly water-soluble or the liquid suspension of amorphous substance.Then, the absorption rate of compound Depending on its rate of dissolution, and rate of dissolution depends on crystal size and crystalline form.Alternatively, by dissolving or suspending compound Realize that delay absorbs the compound of parenteral administration in oily excipient.By in for example poly- friendship of Biodegradable polymeric The storage form of injectable is made in the microcapsules matrix that compound is formed in ester-polyglycolide.According to compound with polymerize The property of the ratio between object and the particular polymer used, controllable produced compounds rate of release.Other Biodegradable polymerics Example includes poly- (ortho esters) and poly- (acid anhydrides).It can also be by the way that compound be trapped in the liposome compatible with bodily tissue or micro- The storage preparation of injectable is prepared in type emulsion.

Per rectum or the composition of vaginal application particularly through mixing compound described herein and are suitble to non-stimulated Property excipient or carrier (such as cupu oil, polyethylene glycol or suppository wax) preparation suppository, the excipient or carrier are in environment At a temperature of be solid but be under body temperature liquid and therefore in rectum or vaginal canal melt and release of active compounds.

Oral dosage form includes capsule, tablet, pill, pulvis and particle.In this solid dosage forms, reactive compound It is mixed at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or Dicalcium Phosphate and/or a) filler Or swelling agent, such as starch, lactose, sucrose, glucose, mannitol and silicic acid, b) adhesive, such as carboxy methyl cellulose, Alginates, gel, polyvinylpyrrolidone, sucrose and Arabic gum, c) moisturizer, for example (,) glycerol, d) disintegrating agent, such as fine jade Rouge -- agar, calcium carbonate, potato or tapioca, alginic acid, certain silicates and sodium carbonate, e) solution retarding agents, such as Paraffin, f) absorbsion accelerator, for example (,) quaternary ammonium compound, g) wetting agent, for example (,) cetanol and glycerin monostearate, h) it absorbs Agent, such as kaolin and bentonite and i) lubricant, such as talcum, calcium stearate, magnesium stearate, solid polyethylene glycol, laurel Base sodium sulphate and its mixture.For capsule, tablet and pill, dosage form also may include buffer.

It can also be used such as lactose or toffee and macromolecule polyethylene glycol excipient by the solid composite of similar type As the filler in soft hard gelatin capsules.Coating and shell, such as enteric coating and the well-known other packets of pharmaceutical field can be used Clothing prepares the solid dosage forms of tablet, lozenge, capsule, pill and particle.They optionally containing opacifiers and can also have group The property of object is closed, so that optionally with delayed mode only discharge active component, or preferably, in certain a part release of enteron aisle. The example of workable embedding composition includes polymer and wax.Lactose or toffee and macromolecule polyethylene glycol etc. can also be used The solid composite of similar type is used as the filler in soft hard gelatin capsules by excipient.

Reactive compound can also be in the microsealing form with one or more above-mentioned excipient.Coating and shell, example can be used As other coatings well-known in enteric coating, controlled release coat and pharmaceutical field prepare tablet, lozenge, capsule, pill and particle Solid dosage forms.In this solid dosage forms, reactive compound may be mixed at least one inert diluent, such as sucrose, cream Sugar or starch.Generally, this dosage form may also comprising other substance besides inert diluents, such as tableting lubricant and Other tabletting adjuvants, such as magnesium stearate and microcrystalline cellulose.For capsule, tablet and pill, dosage form can also Include buffer.They optionally can containing opacifiers and also have the property of composition, so that optionally with delayed mode Only discharge active component, or preferably, in certain a part release of enteron aisle.The example of workable embedding composition includes polymerization Object and wax.

The part of compound described herein or transdermal administration dosage form include ointment, ointment, emulsifiable paste, lotion, gel, pulvis, Solution, spray, inhalant or patch.Aseptically, reactive compound and pharmaceutically acceptable carrier and any need Preservative or the buffer that may need.Ophthalmic preparation, auristillae and eyedrops are also considered within the scope of the present invention. In addition, the present invention considers the purposes with the dermal patch for providing the control attendant advantages that compound is delivered to body.It can lead to It crosses for compound to be dissolved or dispersed in appropriate medium and this dosage form is made.Sorbefacient can also be used for raising compound and pass through The flow of skin.Speed can be controlled by providing rate controlling membranes or by dispersing compound in polymer substrate or gel Rate.

Can also oral, parenteral, applied through part, rectum, nose, oral cavity, vagina or by sucking spray by catheter indwelling With composition as described herein.Term " parenteral " as used herein includes but is not limited to subcutaneous, intravenous, muscle, joint It is interior, synovial membrane is intracavitary, breastbone is interior, intrathecal, liver is interior, intralesional and intracranial injection or infusion techniques.Particularly, in oral, peritonaeum or Intravenous application composition.

The sterile injection form of composition described herein can be water or oil suspension.These suspension can be according to this field Known technology is prepared using suitable dispersion or wetting agent and suspending agent.Sterile injectable preparation is also likely to be can in nontoxic Through the sterile injectable solution or suspension in the external diluent or solvent received of stomach, for example, it is molten in 1,3-BDO Liquid.In acceptable excipient and solvent, adoptable is water, Ringer's solution and isotonic sodium chlorrde solution.In addition, according to Convention is using sterile non-volatile oil as solvent or suspension media.For this purpose, any tasteless fixed oil can be used, Monoglycceride or diglyceride including synthesis.Fatty acid such as oleic acid and its glyceride ester derivatives are used to prepare injection, Because they are natural pharmaceutically acceptable oil, such as olive oil or castor oil, especially it is in polyoxyethylated versions.These Oil solution or suspension may also contain long-chain alcohol diluents or dispersing agent, such as carboxymethyl cellulose or pharmaceutically may be used preparing Common similar dispersing agent in the dosage form (including emulsion with suspension) of receiving.Other conventional surfactants, such as Tweens, Spans and common other emulsifiers or bioavailability in producing pharmaceutically acceptable solid, liquid or other dosage forms Reinforcing agent can also be used for the purpose prepared.

Acceptable dosage form, including but not limited to capsule, tablet, water slurry or solution can be taken orally with any, take orally this Literary described pharmaceutical composition.For for oral tablet, common carrier includes but is not limited to lactose and cornstarch.Allusion quotation Type it is additionally added lubricant, such as magnesium stearate.In order to be applied to capsules per os, useful diluent include lactose and Dried corn starch.When oral application needs water slurry, active constituent is in conjunction with emulsifier and suspending agent.If desired, may be used also Certain sweeteners, corrigent or colorant is added.

Alternatively, pharmaceutical composition as described herein can be applied for the suppository form that rectum uses.Mix reagent can be passed through These pharmaceutical compositions are prepared with non-irritating excipient, the excipient is solid at room temperature, but is under rectal temperature Liquid, therefore will melt in rectum to discharge drug.This substance includes but is not limited to cupu oil, beeswax and polyethylene glycol.

Especially when therapeutic purpose includes that part drop applies easily accessible region or organ, including eye, skin or low level It, can also local application pharmaceutical composition as described herein when intestines problem.It is easy to as each of these regions or organ preparation Suitable topical formulations.

It can be realized with rectal suppository formulation (seeing above) or suitable enema preparation and the part drop of lower bowel is applied.? Local skin patch can be used.

For locally drop is applied, pharmaceutical composition can be formulated as containing suspension or be dissolved in one or more carriers The suitable ointment of active component.Applying the carrier of the compound of the present invention suitable for part drop includes but is not limited to mineral oil, vaseline Oil, albolene, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.Alternatively, pharmaceutical composition can be prepared For containing suspending or be dissolved in the suitable lotion or emulsifiable paste of the active component in one or more pharmaceutically acceptable carriers.It is suitble to Carrier include but is not limited to that mineral oil, sorbitan monostearate, polysorbate60, cetyl esters wax, spermaceti are hard Lipidol, 2- octyl dodecanol, benzyl alcohol and water.

For ophthalmology use, pharmaceutical composition can be formulated as in isotonic pH with or without preservative such as benzalkonium chloride The micronized suspension in Sterile Saline is adjusted, or especially isotonic pH adjusts the solution in Sterile Saline.Alternatively, for ophthalmology It uses, pharmaceutical composition can be formulated as to ointment, such as vaseline.

The spray that can also be gasified by nose or sucking application pharmaceutical composition.According to skill well-known in pharmaceutical field Art prepares this composition and using benzylalcohol or other suitable preservative, the sorbefacient, the carbon that improve bioavailability Fluorine compounds and/or other conventional solubilizer or dispersing agent are prepared into the solution in salt water.

Unit dosage forms can will be configured to the compound.Term " unit dosage forms " refers to the unit dose for being suitable as subject The discrete unit of the physics of amount, per unit contain be computed generate expected effect predetermined amount active material, optionally be suitble to Pharmaceutical carrier combine.Unit dosage forms can make single daily dose or multiple daily dose (for example, daily about 1-4 time or more secondary) It is wherein primary.When using multiple daily dose, the unit dosage forms of each dosage may be the same or different.

V. specific embodiment

Embodiment 1:XRPD, C¹³Solid state NMR, the universal method of DSC measurement

Thermogravimetric analysis (TGA)

Thermogravimetric analysis (TGA) is carried out using TA Instruments TGA model Q500Asset Tag V014840.It will consolidate Body sample is placed in platinum sample disc and is heated to 300 DEG C from room temperature with 10 DEG C/min.

DSC measurement

Differential scanning calorimetry (DSC) is carried out using TA Instruments DSC Q200Asset Tag V015553. About 1-2mg solid sample is put into the sealing DSC disk of the aluminium with the bellows covers with pin hole.Usually add in nitrogen purification atmosphere Hot sample cell.

SSNMR experiment

Use the Bruker-Biospin 400MHz Advance III of installation Bruker-Biospin 4mm HFX probe Wide aperture luminometer obtains the spectrum of solid state nmr spectroscopic assay (SSNMR).Sample is packed into 4mm ZrO₂Rotor (about 70mg or hereinafter, depending on sample utilizability).Typically apply magic angle spin (Magic angle spinning) (MAS) speed is 12.5kHz.Probe temperature is set in 275K so that the frictional heating effect during spin to be reduced to most Lower bound degree.It uses¹H MAS T₁Saturation recovery relaxation experiment measurement proton relaxation time, to set¹³C cross polarization (CP) The recycling appropriate delay of MAS experiment.It will¹³C CPMAS experiment recycling delay adjust at least 1.2 times measure¹H T₁Relaxation time, to maximize carbon spectrum signal to noise ratio.It will¹³The CP time of contact of C CPMAS experiment is set in 2ms.Use band The CP proton pulse of linear ramp voltage (50%-100%).Optimize Hartmann- using external reference sample (glycine) Hahn matching.Fluorine spectrum is obtained using the MAS setting of proton solution idol, wherein recycling delay is set in about 5 times of measurements¹⁹F T₁Relaxation time.Even using proton solution¹⁹F MAS T₁Saturation recovery relaxation measuring fluorine relaxation time.Use SPINAL 64 solutions are even to obtain carbon and fluorine spectrum with about 100kHz field strength.By the gold of its highfield for being set in 29.5ppm of chemical shift reference Rigid alkane external standard.

Bruker D8 Discover XRPD experimental detail

Use the Bruker D8 in installation seal pipe source and Hi-Star area detector (Bruker AXS, Madison, WI) Discover diffractometer (Asset Tag V012842) obtains XRPD pattern in this reflection mode at room temperature.X-ray generator It is operated under 40kV voltage and 35mA electric current.Powder sample is put into aluminium supporter.Respectively two are registered using 120s exposure duration A frame.Then data are integrated within the scope of 4.5 ° of -39 ° of 2 θ with 0.02 ° of step-length and are merged into a continuous pattern.

Embodiment 2: the preparation of the 2-MeTHF solvate of compound (1) and compound (1)

For example, can prepare according to WO 2010/148197 amorphous free alkali compound (1), conventional hand is then carried out Property separation and purifying: using include Et₂NH (generates the Et of compound (1)₂NH salt) modifying agent carry out SCF chiral chromatography, and Then ion exchange resin treatment is carried out.Its XRPD data is as shown in Figure 11.Alternatively, can using the preparation of silica gel following method as The compound (1) of 2-MeTHF solvate:

The preparation of compound 2a (the bromo- 5- fluorine pyridine of 2- amino -3-)

48% hydrobromic acid is added in the slurry in water (24L) to 2- amino-5-fluorine pyridine (6kg, 53.6mol) at 14 DEG C (18.5kg, 110mol) lasts 10 minutes.The exothermic heat of reaction, temperature rise to 24 DEG C.The compound is cooled to 12 DEG C, so After divide 9 parts be added bromine (9kg, 56.3mol), last 50 minutes (heat release is maintained at 20 DEG C).The mixture is stirred at 22 DEG C Overnight, pass through¹(5 drops are quenched into 1ml 20%K to the aliquot of HNMR monitoring quenching₂CO₃, 0.3ml 10%Na₂S₂O₃With The mixture of 0.7ml DCM.Evaporate organic layer, measurement).The mixture is cooled to 10 DEG C, then passes through addition bisulfite Water (2L) solution of sodium (560g, 5.4mol) quenches, and is cooled to 0 DEG C.Add this mixture to cold (- 4 DEG C) DCM In (18L) and 5.4M sodium hydroxide (35L, 189mol) mixture.By Celite pad filtering bottom~35L, then formed Mutually rupture.With DCM (10L) aqueous layer extracted again.Organic layer is filtered by 3kg Magnesol pad, is washed with DCM (8L).Evaporation Filtrate is ground together with hexane, filtering.

Although display 97% is completed in the measurement of method, this initial product run from all 4 times is typical Ground includes~10%SM.Merge them, grind (2L/kg material) in hexane at 50 DEG C, be subsequently cooled to 15 DEG C, filtering obtains To compound 2a (30.0kg ,~95% purity, 149mol, 67%).Color is carried out to the mother liquor from starting grinding and repurity It composes (20kg silica gel, the hexane solution of eluent 25-50%EtOAc), the compound 2a that gets back (4.7kg ,~99% purity, 24.4mol, 11%).

The preparation of compound 3a

Compound 2a (27.5kg, 96% purity, 138mol), Pd (PPh are packed into inertia 400-L reactor₃)₄ (1044g, 0.90mol) and CuI (165g, 0.87mol), is then charged into toluene (90kg).Being given with 3 vacuum-nitrogen circulations should Then triethylamine (19.0kg, 188mol) is added in mixture deoxidation.It is de- to the mixture with vacuum-nitrogen circulation more than once Then TMS- acetylene (16.5kg, 168mol) is added in oxygen.The mixture is heated to 48 DEG C, and (initial heat release needs temperature within 23 hours Degree reaches 53 DEG C of maximum values), it is subsequently cooled to 18 DEG C.Slurry is filtered by Celite pad, is washed with toluene (80kg).With 12% Na₂HPO₄(75L) washs filtrate, is then filtered by silicagel pad (25kg), is washed with 1:1 hexane: MTBE (120L).By the filter Liquid is evaporated to obtain brown oil, is then dissolved in NMP, in next step.Solution weight-the 58kg of compound 3a ,~ 50wt%, 138mol, 100%.¹H NMR(CDCl₃,300MHz):δ7.90(s,1H)；7.33-7.27(m,1H)；4.92(s, NH₂),0.28(s,9H)ppm.

The preparation of compound 4a

Potassium tert-butoxide (17.5kg, 156mol) and NMP (45kg) are packed into inertia 400-L reactor.By the mixture 54 DEG C are heated to, the solution of compound 3a (29kg, 138mol) in NMP (38kg) is then added, lasts 2.75 hours, uses NMP (6kg) rinses (heat release maintains 70-77 DEG C).The reaction system is stirred 2 hours at 74 DEG C, is subsequently cooled to 30 DEG C, The solution of toluene sulfochloride (28.5kg, 150mol) in NMP (30kg) is added, lasts 1.5 hours, is rinsed with NMP (4kg). Exothermic heat of reaction maintains 30-43 DEG C.The reaction system is stirred 1 hour, while being cooled to 20 DEG C, water (220L) then is added, Last 35 minutes (heat release maintains 18-23 DEG C).The mixture is stirred 30 minutes at 20 DEG C, is then filtered, with water (100L) Washing.Solid is filtered out from filter with DCM (250kg), with residual moisture from, by Magnesol (15kg, top) and Silica gel (15kg, lower part) pad filtering organic layer, then washed with DCM (280kg).Concentration filtrate is to obtaining thick slurry (~50L body Product), MTBE (30kg) then is added, while continuing with constant volume distillation (final distillation temperature is 51 DEG C).It adds The slurry is cooled to 15 DEG C by MTBE (10kg), and filtering is washed with MTBE (40L), obtain compound 4a (19.13kg, 95% Purity, 62.6mol, 45%).Partial concentration filtrate obtains second batch (2.55kg, 91% purity, 8.0mol, 6%).¹H NMR (CDCl₃,300MHz):δ8.28-8.27(m,1H)；8.06-8.02(m,2H)；7.77 (d, J=4.0Hz, 1H)；7.54-7.50 (m,1H)；7.28-7.26(m,2H)；6.56 (d, J=4.0Hz, 1H)；2.37(s,3H)ppm.

The preparation of compound 5a

Chemical combination is added in the slurry in DCM (30kg) to N- bromine succinimide (14.16kg, 79.6mol) at 15 DEG C Solution of the object 4a (19.13kg, 95% purity and 2.86kg, 91% purity, 71.6mol) in DCM (115kg), uses DCM (20kg) is rinsed.The mixture is stirred 18 hours at 25 DEG C, 9 DEG C are subsequently cooled to, by adding hypo solution The quenching of (400g) and 50% sodium hydroxide (9.1kg) water (130L) solution.The mixture is warmed to 20 DEG C, separates each layer, is used 12% salt water (40L) washs organic layer.Successively use DCM (4 × 50kg) aqueous layer extracted again.Merge organic layer, distillation 40L and water are total Then boiling filters the solution by silica gel (15kg, bottom) and acid magnesium stearate (15kg, top) pad, with DCM (180kg) Washing.Chlorine is concentrated to thick slurry (~32L volume) is obtained, hexane (15kg) then is added.Hexane (15kg) is being added, simultaneously (52 DEG C of final distillation temperature) is persistently distilled with constant volume.The slurry is cooled to 16 DEG C, filtering is washed with hexane (25kg) It washs, obtains Compound Compound 5a (25.6kg, 69.3mol, 97%).¹H NMR(CDCl₃,300MHz):δ8.34-8.33(m, 1H)；8.07 (d, J=8.2Hz, 2H)；7.85(s,1H)；7.52-7.49(m,1H)；7.32-7.28(m,2H)；2.40(s,3H) ppm.

The preparation of compound 6a: BEFTAI reaction

Compound 5a (25.6kg, 69.3mol), bis- (pinacol combined) two boron are packed into inertia 400-L reactor (19kg, 74.8mol), potassium acetate (19kg, 194mol), acid chloride (156g, 0.69mol) and triphenyl phasphine (564g, 2.15mol), dioxanes (172kg) then is added, uses vacuum-nitrogen circulation difference deoxidation (x 3).The mixture is stirred, is made With vacuum-nitrogen circulation deoxidation (x 2), it is then heated to 100 DEG C 15 hours.The mixture is cooled to 35 DEG C, is then filtered, It is washed with 30 DEG C of THF (75kg).Filtrate is evaporated, residue is dissolved in DCM (~90L).By the solution and 1kg carbon and 2kg acid Magnesium silicate stirs 45 minutes together, is then filtered by silica gel (22kg, lower part) and acid magnesium stearate (10kg, top) pad, It is washed with DCM (160kg).Filtrate is concentrated to thick slurry (~40L volume) is obtained, is then ground at 35 DEG C, hexane is added (26kg).The slurry is cooled to 20 DEG C, filtering is washed with DCM (5.3kg) and hexane (15kg) and then with hexane (15kg), It is dry on filter in nitrogen atmosphere, compound 6a (23.31kg, 56.0mol, 81%) is obtained, is white solid.¹H-NMR Consistent with desired product, HPLC 99.5%, palladium measures 2ppm.¹H NMR(CDCl₃,300MHz):δ8.25(s,1H)；8.18 (s,1H)；8.09-8.02(m,2H)；7.91-7.83(m,1H)；7.30-7.23(m,2H)；2.39(s,3H)；1.38(s,12H) ppm.

The preparation of compound 8a and 9a

Compound 8a: acid anhydrides 7a (24.6kgs, Apex) and quinine (49.2kgs, Buchler) are added in reactor, Then anhydrous PhMe (795.1kgs) is added.Then reactor is cooled to -16 DEG C, to maintain internal reactor temperature < -12 DEG C Rate be added EtOH (anhydrous, 41.4kgs).Maximum temperature for this experimental record is -16 DEG C.Then by the reaction Mixture is in -16 DEG C of stirring 16h.Take out sample, filtering.Drying solid passes through¹H-NMR evaluation, display retain without acid anhydrides. The content of filtration reactor.With PhMe (anhydrous, 20kgs) washing reactor and subsequent wet cake.By obtained solid < 45 DEG C and N₂Pan dryer at least 48h is placed under purging.In this experiment, actual temperature is 44 DEG C, and vacuum is -30inHG. Substance is sampled after 2.5d is dry, 3%PhMe is shown by NMR.After 8hrs, the amt of the PhMe of analysis, which is shown, to be existed Identical 3%PhMe and dry termination.The weight of white solid is 57.7kgs, 76% yield.¹H-NMR shows consistent with structure And chirality SFC is analysis shows that substance > 99%ee.

Compound 9a: quinine salt 8a (57.7kgs) and PhMe (250.5kgs, Aldrich ACS are packed into reactor Grade, > 99.5%), start blender.Content is cooled to < 15 DEG C, is handled and (is handled with the dense HCl of 21.4kgs with 6N HCl 18kgs H₂O), while temperature < 25 DEG C are kept.The mixture is stirred into 40min, visual inspection verifying exists without solid.Stop Stirring settles each phase, separates each phase.PhMe (160kgs is used again；It typically uses as few as possible, calculated value 43kgs) Aqueous phase extracted.However, adding PhMe effectively to stir because of small size.Merge organic phase.Organic phase is sampled, operation HPLC is analysed to ensure that there are products；Only for Test Information.

Sodium sulphate (anhydrous, 53.1kgs) is added into the organic phase for being cooled to < 5 DEG C (0-5 DEG C), stirs 8hrs (this It is 12hrs in situation).Make the content of the reactor comprising organic phase by the inclusion of the filter of sodium sulphate (31kgs, anhydrous), And enter cleaning and dry reactor.Reactor is rinsed with PhMe (57.4kgs), reactor 201 is entered by filter.It opens Dynamic blender, adds a certain amount of PhMe (44kgs), which is cooled to -20 DEG C.At such a temperature, it is added The PhMe solution of tert-pentyl alcohol potassium, lasts 2h, while maintaining the temperature at -15--22 DEG C.The reaction mixture is maintained at about -20 DEG C, using 30min, then sample.By taking out aliquot, it is quenched into 6N HCl at once and is sampled.Target herein Than for 96:4 (trans-: cis-).

Due to obtaining target ratio, so being packed into acetic acid (2.8kgs) into reactor, 6min is lasted.Temperature rests on -20 ℃.Then temperature is adjusted to -5 DEG C, 2N HCL aqueous solution (handling 65.7kgs water with the dense HCl of 15.4kgs) is added.By content Object is warmed to 5 DEG C +/- 5 DEG C, stirs 45min, is then warmed to 20 DEG C +/- 5 DEG C, while stirring 15min.Stop blender, Settle each phase.It takes out water layer (temporarily holding).Organic phase is washed with water (48kgs, potable), stirs 15min, is made each Mutually sedimentation (at least 15min) is taken out water layer, is added in water layer.1/3 buffer solution (50L) of preparation is added into organic phase (7.9kgsNaH₂PO₄、1.3kgs Na₂HPO₄With 143.6kgs water), stir at least 15min.Stop stirring, make each phase separate to Few 15min.Discard lower layer.Another part buffer solution (50L) is for washing organic layer as described above.The is carried out as described above 3 washings.

Start that PhMe phase (150L) is evaporated in vacuo in 42 DEG C/- 13.9psig, distillation is to obtaining about 20L volume grease.? After volume largely reduces, which is transferred to smaller container to complete to distill.It is added heptane (13.7kgs), by the mixture It is warmed to 40+/- 5 DEG C of 30min, content is then cooled to 0-5 DEG C, lasts 1.5h.Filter solid is crossed, with the cold (0-5 of about 14kgs DEG C) heptane wash reactor.It is dried in vacuo solid.The baking oven being so placed in after < 40 DEG C under house vacuum atmosphere (- 28psig) It is < 1% to LOD.15.3kgs, 64%, 96%HPLC purity.¹H NMR(400MHz,CDCl₃)δ11.45(br.s,1H),6.41 (t, J=7.2Hz, 1H), 6.25 (t, J=7.2Hz, 1H), 4.18 (m, 2H), 3.27 (m, 1H), 3.03 (m, 1H), 2.95 (m, 1H), 2.77 (m, 1H), 1.68 (m, 1H), 1.49 (m, 1H), 1.25 (t, J=7.2Hz), 1.12 (m, 1H)

The preparation of compound 10a

To installation mechanical agitator, temp probe, reflux condenser, addition funnel and nitrogen inlet in nitrogen atmosphere Be packed into 3 neck flasks compound 9a (145.0g, 1 equivalent) and dry toluene (Aldrich, catalogue #244511) (1408g, 1655ml).Then by several times into the solution of stirring be added triethylamine (Aldrich, catalog number (Cat.No.) #471283) (140g, 193ml, 2.14 equivalents), 5 minutes are lasted, in the process, observes that heat release to maximum temperature is 27 DEG C.Begin through ReactIR acquisition Data.Then the reaction mixture is heated to 95 DEG C, lasts 70 minutes.Then diphenyl phosphinylidyne is added by several times with addition funnel Base azide (Aldrich, catalogue #178756) (176.2g；138.0ml, 0.99 equivalent), lasting total time is 2.25 small When.

(addition funnel is washed with a small amount of toluene) after the completion of adding diphenyl phosphoryl azide, the mixing that will be obtained Object reheats 50 minutes at 96 DEG C.By the reaction mixture sample of GC/MS analysis dilution with toluene, diphenylphosphoryl is shown Azide exhausts.Then benzylalcohol (Aldrich, catalogue #108006) being added with addition funnel, (69.9g, 67.0ml, 1.0 work as Amount), last 5-10 minutes.Then obtained mixture is heated overnight (about 19 hours) at 97 DEG C.It is shown by GC/MS with dilute The reaction mixture sample released forms product (m/e=330).Then the reaction mixture is cooled to 21 DEG C, hereafter, added by several times Enter water (870g, 870ml) (observing that appropriate heat release to maximum temperature is 22 DEG C).First mix reaction by addition 500g water Object quenching, mechanical stirring 10 minutes.Then the mixture is transferred to the separatory funnel comprising remaining 370g water, then stirred manually It mixes.It is stirring with after mutually separation, is separating organic layer and water layer (cutting water layer in pH~10).Then part water (870g is used again； 1 × 870ml) washing organic layer.Separate organic layer and water layer (cutting water layer in pH~10).Then the organic of acquisition is concentrated under reduced pressure Xiang Zhigan (water-bath, at 45-50 DEG C) obtains 215g crude compound 10a (about 190ml volume).¹H NMR and GC/MS and compound 10a is consistent (containing remaining toluene and benzylalcohol).

The preparation of compound 11a

HCl in ethyl alcohol preparation: it is burnt in nitrogen atmosphere to 3 necks of installation temp probe, nitrogen inlet and magnetic stirrer Ethyl alcohol (1000ml, 773g) is packed into bottle.Stir the solution, be cooled to dry ice/acetone batch reach internal temperature be -12 DEG C. Then making anhydrous HCl (~80g, 2.19moles), slowly blistering (observes temperature in addition observation in cooling solution From -24 to -6 DEG C), last 2 hours.After addition, which is transferred to vial, is warmed to environment temperature.By solution example into Row titration, obtains 2.6M concentration.Then the solution storage is stayed overnight in cold house's temperature (about 5 DEG C).

Hydrogenation/HCl salt is formed: the glass insert dress of 2 gallons of Parr high-pressure reactors is connected in nitrogen atmosphere Enter palladium on carbon (Pd/C (Aldrich, catalogue #330108), 10% butt；(50% is wet), 13.11g, 0.01 equivalent, with compound Based on 10a), with ethyl alcohol (93g；120ml) moisten.Then crude compound 10a (212g, 1eq) is added into glass insert In ethyl alcohol (1246g；Solution (being rinsed on a small quantity with ethyl alcohol to assist being transferred to) in 1600ml).It is anti-that glass insert is put into high pressure Device is answered, the ethanol solution (preparation as described above of HCl is subsequently to added into；2.6M；1.04 equivalents are based on compound 10a；223g； 259ml).High-pressure reactor is sealed, then purifies (3 × 20psi) with hydrogen.Then in the case where hydrogen applies pressure, (15psi) is opened Dynamic hydrogenation 3 hours, at this point, the pressure appearance of hydrogen is constant.Pass through¹The aliquot that H NMR and GC/MS analyze reaction mixture is aobvious Show raw material exhaust/product formed.Then the mixture being obtained by filtration with bed of diatomaceous earth (192g), hereafter, then with ethyl alcohol (3x；? Amounted in washing process and use 1176g ethyl alcohol) washing bed of diatomaceous earth.Then (water-bath, at 45 DEG C) filtrate (green) is concentrated under reduced pressure To~382g (~435ml)；2.9 volumes, the gross data yield based on compound 11a.Then isopropyl acetate (1539g； 1813ml (12 volumes, the theoretical yield based on compound 11a)), it is added in rest part.Vacuum is stepped up using temperature Distill obtained solution.

Stop distillation.Hereafter, make remaining solution (370g ,~365ml total volume；Light brown) in environment temperature standing 1 A weekend.It filters the mixture (isopropyl acetate is used for aided filter), then with isopropyl acetate (2x 116ml；It is each to use by oneself about 100g washing) wash the solid acquired.Then it stays overnight, obtains in 40 DEG C of vacuum drying solids (temperature that highest is observed is 42 DEG C) To 118g (78.1%, 2 steps in) compound 11a.The substance¹H NMR is consistent with the structure of compound 11a and GC/MS is shown 99% purity.

The preparation of compound 13a

Method A: use CH₂Cl₂(169mL) handles the fluoro- 2,4- dichloro pyrimidine of 5- (12a, 39.3g, 235mmol, 1.1 equivalent) With the mixture of HCl amine salt (11a, 50g, 214mmol), which is warmed to 30 DEG C.Then slow by syringe pump Ground handles the mixture with DIEA (60.8g, 82mL, 471mmol, 2.2 equivalent), lasts 3h.Peak temperature reaches 32 DEG C.It should Reaction system stirs 20h, determines that reaction mixture is complete by HPLC, is cooled to rt.Successively with water (211mL, pH=8-9), 5%NaHSO₄The reaction mixing that (211mL, pH=1-2), then 5%NaCl aqueous solution (211mL, pH=5-6) washing obtain Object.

Then organic phase is evaporated under reduced pressure to 190mL.It is packed into PhMe (422mL), temperature is set in 70-80 DEG C and inside Temperature is at 60-65 DEG C, until volume is restored to 190mL.The mixture is cooled to about 37 DEG C, is stirred simultaneously, after about 10min, Crystallization takes place, and observes that temperature increases to about 41 DEG C.After 37 DEG C of balances, n-heptane is added into the suspension (421mL), lasts 3.5h, is subsequently cooled to 22 DEG C, lasts 1h.The mixture is stirred at such a temperature, is then filtered.With The solid obtained on n-heptane solution (2x 210mL) washing nozzle of 10%PhMe.Then in an oven with N₂Under purging 50 DEG C of vacuum drying solid overnights.Obtained solid is weighed as 62g (88% yield).

Method B: to according to mechanical agitator, temp probe, reflux condenser, nitrogen inlet and liquid feeding in nitrogen atmosphere Compound 11a (51.2g) and compound 12a (40.2g) are packed into 3 neck flasks of funnel.Addition methylene chloride (173ml, 230g), the mixture stirred to get, while being warmed to internal temperature is 30 DEG C.Then N, N- are slowly added into addition funnel Diisopropylethylamine (85ml, 63.09g) lasts 2.5-3 hours, and in the process, heat release to the maximum temperature observed is 33.5℃.After the addition was complete, obtained solution is stirred overnight (about 19 hours) at 30-31 DEG C in nitrogen atmosphere.

It is 10ml that 100 μ l samples of the reaction mixture, which are diluted to total volume, with methylene chloride, is sufficiently mixed the solution. The sample that dilution aliquot is analyzed by GC/MS shows that reaction is completed by GC/MS；Observe that product forms (m/e= 328)).The reaction mixture is cooled to 26 DEG C, is transferred to separatory funnel (being assisted with methylene chloride).Then water is successively used (211ml,211g；The pH for cutting water phase is~8；The layer (raglayer) cut on a small quantity is shifted with the water layer that cuts), 5% NaHSO₄Aqueous solution ((being prepared using 50g sodium bisulfate monobydrate (Aldrich catalogue #233714) and 950g water) 211ml, 216g；The pH for cutting water layer is~2), then prepared with (using 50g sodium chloride (Aldrich catalogue #S9888) and 950g water) 5%NaCl aqueous solution (211ml, 215g；The pH of the water layer cut is~4-5) wash the mixture.Then it is concentrated under reduced pressure and adopts To~190ml, (hereafter based on the theoretical yield of compound 13a, first is added in 2.7 volumes to the organic phase (water-bath, at 35 DEG C) of collection Benzene (Aldrich catalogue #179418,422ml, 361g).The mixture (water-bath, at 55-65 DEG C) being concentrated under reduced pressure to give to~ 190ml (2.7 volumes, the theoretical yield based on compound 13a).Pass through at this stage¹H NMR analytical solution sample, which is shown, not to be deposited In methylene chloride.Remaining mixture is cooled to 37 DEG C (using water-bath, at 37 DEG C, with rotary evaporations and stirring).It crosses herein Cheng Zhong observes significant crystallization.Then mechanical stirring mixture is heated to about 37 DEG C (external heat source is set to 38 DEG C), Hereafter, n-heptane (430ml, 288g are slowly added by addition funnel；Aldrich catalogue #H2198), last 3 hours.? After addition, stop heating, the slurry that mechanical stirring obtains, while being cooled to ambient temperature overnight.Then the mixing being obtained by filtration Object, with n-heptane solution (2 × 210ml of 10% toluene；It is n- each by mixing 21ml (16g) toluene and 189ml (132g) Heptane prepares cleaning solution) wash the solid acquired.Apply vacuum, until almost no longer observing filtrate.Then at 50 DEG C in nitrogen It is further dried in vacuo solid in air-flow atmosphere to constant weight (3.5 hours), obtains 64.7g (90%) compound 13a.Pass through¹H NMR to solid sample carry out analysis shows that the substance with structure consistent and LC analysis shows that being using the purity of the LC method of offer 99.8%.

The preparation of compound 14a

Ethyl ester 13a (85g, 259mmol) is dissolved in THF (340mL), at LiOH solution (2M, 389mL, 778mmol) Reason, lasts 10min (from 21 to 24 DEG C of temperature).The mixture is warmed to 45 DEG C, while stirring 17h, at this point, being sentenced by HPLC (not observing SM) is completed in fixed reaction.The reaction mixture is cooled to rt, CH is added₂Cl₂(425mL).Then it is slowly added into Citric acid solution (2M, 400mL) lasts 45min (temperature reaches 26 DEG C).Note that finding that some whites are solid in adding procedure Body, but quickly dissolution under stiring.The reaction mixture is stirred for 15min, then separates each phase.After the separation of each phase, The pH for measuring water phase is pH=4.0.Organic phase (15min stirring) is washed with water (255mL), separates each phase.Then will include Lower layer's (organic layer) of desired product is stored in refrigerating box overnight.

It is concentrated under reduced pressure organic phase (tank is set to 65 DEG C) to about 150mL (est.1.76vol wrtSM).IPA is added (510mL), vacuum distillation (85 DEG C of chiller temperature settings) to 255mL (3vol).Make aqueous solvent by adding IPA (298mL) It is flat to reach about 553mL (6.5vol).Then water (16mL) is added, which is warmed to reflux (77 DEG C), while good Good stirring, the solid dissolution precipitated on the wall.Then reaction mixture is slowly cooled to 65 DEG C (lasting 60min), Holding-total material (takes out sample to analyze for residual solvent) still in the solution.The reaction system is cooled to 60 DEG C, It is opaque that appropriateness occurs in the reaction mixture.After stirring 15min, it is cooled to 55 DEG C.Although more product precipitatings, this is mixed Conjunction object is still thin and is easy to stir.Pole is slowly added into water (808mL) (2.5-3hrs), while maintaining temperature is about 55 DEG C. Then the mixture is cooled to 22 DEG C, lasts 2h, be stirred overnight.Then filter material, with water: the mixture of IPA (75:25, 2x 255mL) washing.Acid is dried overnight in vacuum drying oven at 55 DEG C.Obtain 69g acid 14a, 88% yield, white solid.It is logical Cross the substance > 99% purity of HPLC analysis.

The preparation of compound 15a: Suzuki coupling

To 14a (91.4g, 305mmol), 6a (158.6g, 381mmol, 1.25 equivalent), Pd (OAc)₂(0.34g, 1.5mmol, 0.5mol%), X-Phos (1.45g, 3.0mmol, 1.0mol%) and K₂CO₃(168.6g, 1220mmol, 4 work as Measure) in THF (731mL, 8 volumes) and water (29mL, 0.32vol) is added.Use N₂Reaction mixture 30min is purged, then It is warmed to 65-70 DEG C, stirs 5h.The HPLC of the reaction mixture is analysis shows that 99.3% conversion ratio.The reaction mixture is cold But to 22-25 DEG C, water is added.The mixture is stirred, each phase is separated, decants water phase.18wt%NaCl is added into organic phase Aqueous solution (semi-saturation NaCl aqueous solution), is adjusted the pH of the mixture to 6.0-6.5 using 2N HCl.Each phase is separated, is strained Bleed phase.Organic phase is concentrated to small size, acetonitrile is added.It repeats the process 1 time, addition acetonitrile to final volume to 910mL (10vol).The slurry is warmed to 80-85 DEG C of 6h, is subsequently cooled to 20-25 DEG C.The slurry is stirred into 2h, is then filtered.With Acetonitrile rinses solid, obtains 15a (161g, 89% yield).

The preparation of compound (1): detosylated step

THF (125ml, 5vol) is added in 15a (25g, 45.2mmol), be then added MP-TMT resin (6.25g, 25wt%).By the mixture in 20-25 DEG C of stirring 16h, filtering is rinsed with 1vol THF.Repeat resin treatment process and mistake Filter.THF solution is concentrated into 5vol.2M LiOH aqueous solution (90.3mL, 4 equivalents) are added into the mixture at 22-25 DEG C. The reaction mixture is warmed to 40-45 DEG C, stirs 5h.HPLC is analysis shows that 99.7% conversion ratio.The reaction mixture is cold But it to 22-25 DEG C, is added MTBE (50mL, 2vol).Generation mutually separates.Acquire the water phase of lower part.With MTBE aqueous phase extracted.Acquisition The water phase of lower part.2-MeTHF is added into water phase, stirs the mixture.The pH of the mixture is adjusted to 6.0-6.5, decantation Lower aqueous.Organic phase is washed with 6.5 safflower herbal medicine of pH.Organic phase is concentrated into 85mL, is diluted with 2-MeTHF (150mL), It is concentrated into 180mL final volume.Obtained slurry is warmed to 70-75 DEG C, stirs to being completely dissolved, is subsequently cooled to 45-50 DEG C, obtain slurry.Slurry is stirred into 1h, heptane (180mL) then is added.The slurry is cooled to 20-25 DEG C, 1h is lasted, stirs Mix 16h.The batch is filtered, rinses solid with heptane.Drying solid obtains crude compound (1) 2-MeTHF solvate, 79% yield.

Embodiment 3: the formation of the polymorph of the HCl salt of compound (1)

3A: the HCl salt 1/2H of compound (1)₂The preparation of the form A of O

By in the mixture of water and organic solvent the 2- methyltetrahydrofuran (2-MeTHF) of mixed compound (1) it is molten The HCl salt 1/2H of agent conjunction object (1 equivalent) (compound (1) 1 (2-MeTHF)) and hydrogen chloride prepare compound (1)₂The shape of O Formula A, wherein the mixture of water and organic solvent has the water activity of 0.05-0.85.Specific reaction condition used is summarised in In the following table 1:

Table 1: it is used to prepare the HCl salt 1/2H of compound (1)₂The reaction condition of the form A of O

* 2 steps: then iPrOH/AcOH is stirred for into slurry in acetone/water.

Alternatively, also passing through the HCl salt 1/2H of following method prepare compound (1)₂The form A of O:Method A: by compound (1) 2-MeTHF (953g, 2.39mol) is put into the jacketed reactor of 30L, is handled with IPA (15L) and water (0.57L).It opens The reaction mixture is warmed to 73 DEG C by blender, so that every kind of substance is formed solution, is subsequently cooled to 50-55 DEG C.In 50-55 DEG C, the reaction mixture is handled by being slowly added with the IPA solution (0.83M, 4.34L) of freshly prepared HCl, lasts 4h. It should be noted that the point at about 1/2, mixture start retrogradation.Reaction system is sampled, checks correct shape will pass through XRPD Formula.After addition, making cooler according to program, speed change lasts 480min to 0 DEG C under stiring.Confirmation form is analyzed by XRPD Afterwards, slurry is divided into two filters.With 3L IPA washing reactor, for autoreactor IPA flushing liquor~1.5L IPA Wash every kind of filter cake.Filter cake use is vacuumized into air dried overnight.Then filter cake is put into pan dryer, in vacuum and N₂Purification In atmosphere (22inHg) for 24 hours, no heating.Residual solvent and water are analysis shows that 505ppm IPA, 8ppm 2-Me-THF peace treaty 2.15%H₂O.Substance is taken out from baking oven, is co-mulled and made into the group of smashing fastly, is obtained the HCl salt 1/2H of 805g compound (1)₂O。 Method B: alternatively, substituting IPA using acetone, but carrying out according to the mode similar with method A as described above, forms compound (1) HCl salt 1/2H₂O。

The HCl salt 1/2H of compound (1)₂The XRPD and C of the form A of O¹³SSNMR data difference is as shown in figs. 1 and 2. By some peaks XRPD observed and C¹³The peak SSNMR is summarised in respectively in table 2 and 3.

Table 2: the HCl salt 1/2H of compound (1)₂The peak XRPD of the form A of O

The peak XRPD	Angle (θ ± 0.2 2-)	Intensity %
			1	10.5	100.0
2	5.2	71.6
			3	7.4	46.8
4	18.9	42.0
			5	25.2	41.7
6	16.5	39.5
			7	18.1	28.1
8	23.0	27.5
			9	24.1	25.3
10	20.2	21.6
			11	26.4	21.3
12	15.8	19.8
			13	21.8	18.3
14	13.8	17.6
			15	27.4	17.3
16	29.0	16.7
			17	14.8	15.0
18	32.0	15.0
			19	25.7	13.8
20	28.6	13.4
			21	33.8	13.0
22	12.8	12.0
			23	30.8	11.7
24	32.4	11.6
			25	24.5	11.5
26	23.4	11.1
			27	21.0	10.4

Table 3: the HCl salt 1/2H of compound (1)₂The C of the form A of O¹³The peak SSNMR

Peak #	Chemical shift [± 3ppm]	Intensity [rel]
			1	180.1	50.4
2	157.9	9.1
			3	154.6	26.4
4	150.7	25.3
			5	144.9	31.0
6	140.1	6.7
			7	132.4	36.3
8	131.2	30.0
			9	129.0	21.0
10	117.5	33.6
			11	114.0	38.0
12	107.0	34.4
			13	54.8	42.0
14	47.7	52.7
			15	29.2	100.0
16	24.6	74.0
			17	22.1	83.6

It was found that the HCl salt 1/2H of the compound (1) of preparation₂The form A of O be in following solvent system it is stable (but It is not limited to): chlorobenzene, hexamethylene, 1,2- dichloroethanes, methylene chloride, 1,2- dimethoxy-ethane, hexane, 2-methyl cellosolve, Methyl butyl ketone, hexahydrotoluene, nitromethane, naphthane, dimethylbenzene, toluene, 1,1,2- trichloroethanes, acetone, anisole, N-butyl alcohol, 2- butanol, butyl acetate, t-butyl methyl ether, cumene, ethyl alcohol, ethyl acetate, ether, Ethyl formate, heptane, second Sour isobutyl ester, isopropyl acetate, methyl acetate, 3- methyl-1-butanol, methyl ethyl ketone, 2- methyl-1-propyl alcohol, pentane, 1- third Alcohol, 1- amylalcohol, 2- propyl alcohol, propyl acetate, tetrahydrofuran, methyltetrahydrofuran.

Specifically, for the HCl salt 1/2H of compound (1)₂The solubility and stability test of the form A of O, makes chemical combination The sample of object and 500 μ l solvents are loaded into 2mL HPLC bottle.The mixture is stirred 2 weeks in environment temperature, then passes through centrifugation Filtering, the solid analyzed by XRPD, by quantitative NMR to the solubility of quinhydrones standard items analytical solution.Result is general It includes in table 4.

Table 4: the form and dissolubility data of the form A of the HCl salt of compound (1) is summarized

By the way that sample is put into platinum sample disc and obtains thermogram by being heated to 300 DEG C from room temperature with 10 DEG C/min Data (data are not shown).Thermogram data show that 30 ° of -170 DEG C of weight savings are 2.1%, with semihydrate theoretical value (2.0%) consistent.

Sample is heated to 300 DEG C from room temperature by 10 DEG C/min and obtains DSC thermogram data (data are not shown). DSC thermogram data show 50-100 DEG C of dehydration start-up temperature, followed by 200-260 DEG C starts fusing/decomposition temperature.

3B: the HCl salt 3H of compound (1)₂The preparation of the form D of O

By by the HCl salt 1/2H of compound (1)₂The form A of O (has in iso- third alcohol and water or acetone and water or water Water activity equal to or more than 0.9) in stir into the HCl salt 3H of slurry prepare compound (1)₂The form D of O.

For example, by the HCl salt 1/2H of 100mg compound (1)₂The iso- propyl alcohol that the form A of O is 0.9 in 5mL water activity/ Slurry in water or acetone/water is stirred overnight in environment temperature.Supernatant is decanted, obtained solid matter is moderately air-dried, is obtained To the HCl salt 3H of compound (1)₂The form D solid matter of O.

The HCl salt 3H of compound (1)₂The XRPD and C of the form D of O¹³SSNMR data difference is as shown in Figures 3 and 4.It will Some peaks XRPD observed and C¹³The peak SSNMR is summarised in respectively in table 5 and 6.

Table 5: the HCl salt 3H of compound (1)₂The peak XRPD of the F of the form of O

Table 6: the HCl salt 3H of compound (1)₂The C of the form D of O¹³The peak SSNMR

Obtain MDSC thermogram data by the way that sample is heated to 350 DEG C from -20 DEG C with 2 DEG C/min (data are not shown) And ± 1 DEG C was adjusted every 60 seconds.MDSC thermogram data show lower than 150 DEG C dehydrations, melt and at 150 DEG C -200 DEG C Recrystallization, and it is higher than 250 DEG C of degradations.

Thermogravimetric analysis (TGA) also is carried out to the form.Thermogram shows at most 125 DEG C of weight savings 12%, close to three Hydrate theoretical value (11%).It shows that second step is lower than 200 DEG C of weight saving by TGA-MS, is the loss of HCl.Fusing/ It decomposes and opens at about 270-290 DEG C.

3C: the preparation of the form D of compound (1) HCl salt

HCl salt 1/2H generally by making compound (1)₂The nothing of the HCl salt of form A dehydration prepare compound (1) of O Water form D.Dehydration can be carried out by the combination of heating or the purification of dry nitrogen or both.For example, heating 2mg compound with hot plate (1) HCl salt 1/2H₂The form A of O generates desired anhydrous form D at about 85 DEG C.

The XRPD and C of the anhydrous form D of the HCl salt of compound (1)¹³SSNMR data difference is as shown in Figures 5 and 6.By one The peak XRPD and C observed a bit¹³The peak SSNMR is summarised in respectively in table 7 and 8.

Table 7: the peak XRPD of the form D of the anhydrous HCl salt of compound (1)

The peak XRPD	Angle (θ ± 0.2 2-)	Intensity %
			1	5.3	100.0
2	10.5	56.0
			3	15.9	49.2
4	25.9	30.5
			5	21.0	24.6
6	26.5	24.1
			7	5.8	22.6
8	7.4	21.7
			9	19.0	17.4
10	16.6	17.2
			11	25.3	16.1
12	24.7	16.0
			13	29.4	15.5
14	13.8	14.6
			15	20.3	14.5
16	32.0	14.4
			17	19.5	12.4
18	28.6	12.4
			19	17.1	11.5
20	30.3	11.4
			21	27.5	11.0
22	27.0	10.7
			23	23.7	10.4
24	28.0	10.2
			25	21.6	10.1

Table 8: the C of the form D of the anhydrous HCl salt of compound (1)¹³The peak SSNMR

3D: water activity test

With water activity be 0.0-0.8 isopropanol/water compound (1) HCl salt 3H₂The chemical combination of the form D kind crystalline substance of O The HCl salt 1/2H of object (1)₂The competitive slurry studies have shown that of the form A of O after stir about 2 weeks, is being changed at ambient conditions Close the form D of the anhydrous HCl salt of object (1), the HCl salt 3H of compound (1)₂The HCl salt 1/ of the form D and compound (1) of O 2H₂In the form A of O, form A is most stable form.When IPA/ water activity is 0.9, the HCl salt 1/2H of compound (1)₂O's Form A is converted to the HCl salt 3H of compound (1)₂The form D of O.The result studied from these is summarised in the following table 9.

Table 9: to the HCl salt 1/2H of the compound (1) in IPA/ aqueous mixtures₂The water activity of O is tested

The HCl salt 3H of the form D (" form D ") of anhydrous HCl salt compound (1), compound (1)₂The form D (" shape of O Formula F) and compound (1) HCl salt 1/2H₂Phasor of the temperature to water activity in variation between the form A (" form A ") of O As shown in Figure 12.

3F: the unbodied HCl salt of compound (1)

By the Me for handling compound (1) with 1.05eq.NaOH in water and 2-MeTHF₂NEt salt (1.985g) formationization The unbodied HCl salt of object (1) is closed, is then handled with HCl to remove amine and be precipitated from water layer (pH 2-3).It is concentrated to get Slurry to remove any organic matter, then filter.The solid rinsed with fraction water, it is dry.According to WO 2010/ The Me of 148197 prepare compounds (1)₂Then NEt salt carries out conventional chiral separation and purifying: SCF chiral chromatography, use Including Me₂The modifying agent of NEt (generates the Me of compound (1)₂NEt salt).

Embodiment 4: the formation of the polymorph of free alkali compound (1)

4A: the preparation of the form A of free alkali compound (1)

The A (i.e. the form A of compound (1)) by way of following method produces free alkali compound (1): will be thick amorphous Free alkali compound (1) (about 135g) is transferred to the jacketed reactor of 4L, and ethyl alcohol (2.67L) and water are packed into reactor (0.325L) (10% aqueous solution).The mixture is heated to flowing back.Water is added into the mixture that step 2) obtains 20% aqueous solution is made in (300mL).Then obtained mixture is cooled to 55 DEG C (rate=- 1 DEG C/min), then kept 30 minutes.The crystal seed (1.5g, 3.756mmol) of the form A of compound (1) free alkali is then added to cooling mixture, it will Obtained mixture is kept for 30 minutes, while product precipitates.By by amorphous free alkali compound (1) (20mg) in nitro first The crystal seed of the form A of slurry production compound (1) free alkali is stirred into alkane (0.5mL).By by amorphous free alkali chemical combination Object (1) (900mg) stirs into slurry together with the crystal seed for using nitromethane to obtain in acetonitrile (10mL) and produces other change Close the seed crystal material of the form A of object (1) free alkali.Slowly into the mixture of the form A crystal seed comprising compound (1) free alkali Water (795.0mL) is added in ground, and 40% aqueous solution is made.The cooling of obtained mixture is slowly cooled to 0 DEG C (~-10 DEG C/small When), then kept for 2 hours.Then solid matter is filtered, is air-dried, is then air-dried 18 hours again in an oven at 60 DEG C.

Alternatively, the 2- methyl THF solvate using free alkali compound (1) substitutes amorphous free alkali compound (1), Similar fashion also obtains the form A of free alkali compound (1) as described above.

It was found that the form A of the compound (1) of preparation is stable (but being not limited to): acetonitrile, chlorine in following solvent system Benzene, chloroform, hexamethylene, 1,2- dichloroethanes, methylene chloride, 1,2- dimethoxy-ethane, ethylene glycol, formamide, hexane, methyl Butyl ketone, hexahydrotoluene, N-Methyl pyrrolidone, nitromethane, naphthane, toluene, 1,1,2- trichloroethanes, acetic acid, fennel Fragrant ether, n-butyl alcohol, butyl acetate, cumene, ethyl acetate, ether, Ethyl formate, heptane, isobutyl acetate, isopropyl acetate, 3- methyl-1-butanol, 2- methyl-1-propyl alcohol, pentane, propyl acetate, water, the iso- propyl alcohol of water-(1:3 volume/volume) and water-second Nitrile (1:1 volume/volume；1:3 volume/volume).

The XRPD and C of the form A of compound (1)¹³SSNMR data are respectively as shown in Fig. 7 and 8.Respectively by some observations The peak XRPD and C arrived¹³The peak SSNMR is summarised in table 10 and 11.

Table 10: the peak XRPD of the form A of compound (1)

Table 11: the C of the form A of compound (1)¹³The peak SSNMR

Thermogravimetric analysis (data are carried out using form A of the TA Instruments TGA model Q500 to product Compound (1) Do not show herein), the disk is heated to from room temperature by the way that its sample to be placed in platinum sample disc and then with 10 DEG C/min 300 DEG C carry out.Thermogram display, which is decomposed, to be started at about 293 DEG C.

The DSC thermogram of the form A of compound (1) is also obtained using TA Instruments DSC Q200.By the shape The sample of formula is heated to 350 DEG C with 10 DEG C/min.DSC thermogram shows that fusion temperature is about 278 DEG C.

4B: the preparation of the form B of the hydrate of free alkali compound (1)

The hydrated form of free alkali compound (1) shows that with the form A of free alkali compound (1) be the isomorphism.Work as exposure When high humility, the form A of free alkali compound (1) can freely be converted to hydrated form B, when humidity reduces, restore. According to the phase transformation (data are not shown) for using DSC measuring, transition temperature is close to environment temperature and with the difference of water activity And change.For example, at ambient temperature, observing hydrated form, wherein water activity is greater than 0.6, such as 0.6-1.0.

4C: the preparation of amorphous free alkali compound (1)

Suzuki coupling is by by chlorine pyrimidine, compound 13a, boric acid ester compound 6a, catalyst Pd (OAc)₂And ligand (X-Phos) 10vol 2-MeTHF is dissolved in be coupled.The mixture is heated to 65 DEG C, to maintain reaction mixture at 65 DEG C Rate be added 2vol 50%K₃PO₄Aqueous solution.Two fully reacting conversions, are subsequently cooled to 20 DEG C, are filtered by diatomite. Water layer is separated, discards, washs organic layer with 5%NaCl aqueous solution, so or is concentrated to dryness, about 3.5kg bottle green paste is respectively obtained Shape object.Thick grease is divided into 4 equal portions, with 400g SiO₂Slurry is stirred into together with 500g Florisil, passes through 2.3kg SiO₂Column and heptane/EtOAc (5:1-3:1,2L fraction) elute, and merge the fraction comprising whole products.These fractions are concentrated To doing, about 2.9kg compound 21a is obtained.

Compound 21a is dissolved in 10vol (25L) CH₃CN, with 4eq.HCl (Isosorbide-5-Nitrae-dioxane of 4.31L4N HCl) In 70 DEG C of processing 15h.Determine that reaction 100% is completed by HPLC.Thin slurry is cooled to 20 DEG C, lasts 1h.With 0.5L/ TBME (28L, 11vol) is added in min, and slurry becomes extremely thick (gel) at the end of addition.After 4-5h stirring, the silt Slurry becomes very thin.By filtering the solid collected, is washed with 3 × 5L TBME, low-density filter cake is obtained, in N₂Stream It is 3 days dry in atmosphere, obtain the compound 22aHCl of 1.71kg (86% yield, 98.9%AUC purity).

20 DEG C by NaOH solution (55.60mL 2M, 111.2mmol) be added to compound 22aHCl (10g, 22.23mmol) in the suspension in 2-MeTHF (100.00mL).By the reaction mixture in 60 DEG C of stirring 5h, then exist again 67 DEG C of stirrings.After stirring in about 22 hours, in mixture that 100mL (10vol) 2-MeTHF is added to.Then by this batch Amount is cooled to 0 DEG C.HCl is added into obtained mixture to adjust pH to pH6.6, generates thick free alkali compound (1). Thick material in 60mL (6vol) 2-Me-THF is heated to 50 DEG C.50mL (5vol) n-heptane is added to mixed It closes in object, lasts 1 hour.Then this is cooled to 20 DEG C in batches.Solid product is filtered, then passes through column chromatography (EtOAc/ heptan Alkane 2:1-4:1) pure solid product.Its XRPD data shows amorphous free alkali compound (1).

Alternatively, in the form A of free alkali compound (1) and selected from cellosolvo, 2-methyl cellosolve, tert-butyl first Observe amorphous free alkali compound (1) (for example, with reference to following table in the solvent mixture of base ether, formic acid or methyl ethyl ketone 13) it, stirs at ambient temperature.

4D: the preparation of the 2-MeTHF solvate of free alkali compound (1)

The prepare compound (1) 1 (2-MeTHF) as described in above-described embodiment 2.Its XRPD data is as shown in Figure 10.It will Its XRPD data summarization is in table 12.

Table 12: the peak XRPD of compound (1) 1 (2-MeTHF)

The peak XRPD	Angle (θ ± 0.2 2-)	Intensity %
			1	6.4	9.78
2	8.4	38.07
			3	9.7	43.96
4	12.9	15.57
			5	16.7	100
6	16.9	46.55
			7	17.4	18.67
8	19.4	16.54
			9	20.0	14.62
10	21.0	20.4
			11	21.3	13.58
12	22.3	37.59
			13	24.3	15.36
14	25.7	16.34
			15	25.9	10.06

4F: the form A and amorphous compound (1) of free alkali compound (1) solubility in different solvents system and Stability data

Trip is tested according to the mode similar for the form A of HCl salt of compound (1) as described above at ambient temperature From the dissolution of alkali cpd (1) form A (" form A ") and amorphous compound (1) (" amorphous ") in different solvents system Degree and stability.By obtained data summarization in table 13.

Table 13: the dissolution of form A (" the form A ") and amorphous compound (1) (" amorphous ") of free alkali compound (1) Degree and stability data

Embodiment 5: the preparation of the form A of the toluene fulfonate of compound (1)

The form A of the toluene fulfonate of compound (1) is by by amorphous free alkali compound (1) (500mg) and p- first Benzene sulfonic acid stirs into slurry preparation in acetonitrile (20ml).Sample is stirred overnight.Its XRPD data is as shown in Figure 9.By one The peak XRPD for the compound observed a bit is summarised in table 14.

Alternatively, can according to similar mode as described above, use the 2- methyl THF solvent of free alkali compound (1) It closes object and substitutes amorphous free alkali compound (1) A in the form of the toluene fulfonate of prepare compound (1).

Table 14: the peak XRPD of the form A of the toluene fulfonate of compound (1)

Embodiment 6: the preparation of compound (1)

A. the tablet of compound (1)

Composition

The HCl salt 1/2H of compound (1)₂The form A (the hereinafter referred to as compound (1) of embodiment 6) of O is used for tablet It is formed.Whole excipient meet the newest monograph standard of European Pharmacopoeia (European Pharmacopoeia) and USP/NF and Purchased from approved supplier.

For the preparation of prefabricated grain blend and granulation binders solution composition and batch size as shown in table 15A.It is viscous The batch size of mixture solution includes 100% excessive for calibration of pump and starting solution pipeline.Theory compacting blend composition As shown in table 15A.Yield based on dry particle calculates the actual amount for being used for the batch.The composition of film coating suspension It is as shown in table 15B and excessive for calibration of pump and starting suspension pipeline including 100% with approximate batch size.Film coating The target amount of layer accounts for the 3.0%w/w of tablet weight.

Table 15A: the composition of the tablet of compound (1)

Table 15B: film-coating suspension composition and approximate batch size

Binder solution preparation

Binder solution is made of povidone and water.Water content based in final particle 40% prepares the solution.Therefore, The total amount (povidone) of solid in the solution is 3.6% (w/w).Preparation 100% is excessive for starting pipeline etc..It is tried based on particle The visual inspection for testing starting prepares the other stock solution of +/- 2% (38-42%) water in final particle.Typically, it weighs Under constant stirring the container comprising DI water is added in PVP K30 by 87.00g PVP K30 and pure (DI) water of 2320.00g. After addition, sealing container minimize to evaporate, which is stirred to existing all solids and is completely dissolved.

Wet granulation process

Wet granulation is carried out by following methods: by compound (1), AvicelPH-101, the Fastflo of excessive (10%) Lactose and croscarmellose sodium weighing (referring to table 15A).They are sieved or installed firmly 813 μm using 20 mesh and rubs broken sieve mesh with the hands Cone mill (U5Quadro Co-mill) be sieved with 1000rpm.The substance of sieving is put into single sack or container.Then These substances are transferred to blender, typically with 15rpm compounding 15 minutes.Use the U5Quadro taper for having 4mm square hole screen Mill is with the substance of 1000rpm grinding compounding.It is compounded the substance of grinding again, repeats blending step.Then the substance that will be compounded again It is fed into twin-screw granulator.Batch wet granular is fed into granulation using loss in weight feeder (K-tron or the like) Machine.Then obtained substance is pelletized.Adhesive fluid (referring to table 15A) is injected into twin-screw granulator using peristaltic pump.It is molten The ratio between liquid feed rate and powder feed rate are 0.4095.For example, if powder feed rate is 15.00g/min, solution Feed rate is 0.4095*15.00=6.14g/min, and wherein water content is 40% (being based on dry mass).Particle batch is adopted Collect the basin into preparatory taring.The substance of acquisition is equably sprayed on disk, with the oven drying substance, forms dry Grain.Dry particle is put into K-tron and is fed into cone mill in a manner of continuously grinding, is then ground.

Match mixing pressing process outside particle

It carries out matching mixing pressing process outside particle by following method: the extra-granular based on compacting blend composition of weighing The amount of shape agent.The excipient of weighing is sieved with 1000rpm using the U5Comil with 32C sieve and circular rod impeller.First will Compound (1) particle of grinding is added in the blender of Avicel PH-102 and Ac-Di-Sol comprising sieving.By they Compounding was with 16RPM8 minutes.Sodium stearyl (SSF) is sieved into suitable container by 50 mesh firmly.SSF mass will be approximately equal to The outer blend of 10 times of partial particulate and SSF be put into container, bag is compounded 30 seconds, then adds the mixture to box stirring Machine.Then by total material with 16rpm compounding 2 minutes.Then according to the prepared final compounding of tablet press machined parameters compacting Object.

Film coating

Using based calcium as the white #33G water of 15%w/w Opadry II in the flat coating pan of Vector VPC 1355 Property suspension is applied on label.Target coatings are the 3.0%w/w plate core weight with 2.5%-3.5% tolerance interval.For Reach this target, be equivalent to the amount of the increased coating suspensions of 3.2% weight by spraying, obtains 3.0% coatings, presumption packet Clothing efficiency is 95%.

B. intravenous (IV) preparation of compound (1)

By the HCl salt 1/2H of compound (1)₂The form A (the hereinafter referred to as compound (1) of embodiment 6) of O is as use It is provided in the 2mg/mL solution of intravenous (IV) application.The composition of the solution and the function of quality reference and every kind of ingredient are provided In table 16A and 16B.

Table 16A: the composition of solution excipient^a.

^aWith the pH of NaOH or HCl adjustment solution

Table 16B: the composition of compound (1) intravenous solution^a.

^aWith the pH of NaOH or HCl adjustment solution.Solution density is 1.000g/cm³。

^bDrug substance is semihydrate HCl salt.The amount of drug substance is calculated based on inactive anhydrous free base equivalents, wherein The transforming factor of free alkali and semihydrate HCl salt is 1.11.

Embodiment 7: the in vivoassay that compound (1) is combined with or without Oseltamivir

With excipient or the HCl salt 1/2H for the compound (1) for enhancing dosage level₂O form A and clinically relevant dosage Infecting mouse is treated in combination in Oseltamivir, after 48 hours A type influenza challenges or before Type B influenza challenge 2 hours.

Method: in these researchs, with the figuration comprising 0.5% (w/v) MC (Sigma-Aldrich, StLouis, MO) The form A (the hereinafter referred to as compound (1) of embodiment 7) of the HCl salt semihydrate of compound (1) is prepared in agent, is obtained uniformly Suspension, and compound dosage is based on the HCl salt semihydrate of compound (1).The deionized water of distillation prepares Oseltamivir, obtains To homogenous suspension.With the combination of excipient compound (1) and Oseltamivir comprising 0.5% (w/v) MC.It is grinding every time Combination preparation is prepared when studying carefully beginning and is stored at 4 DEG C at most 10 days, while being stirred in the dark.By whole preparations and excipient Mouse is applied to the applied volume of 10mL/kg by oral cavity gavage.

Influenza virus A/the PR/8/34 or B/Mass/3/66 adapted to the mouse of lethal dose passes through anesthesia of intranasally instiling With inoculation male Balb/c mouse (5-7 weeks, 17-19 grams).8 mouse are registered by each study group.Treatment is after the inoculation of A type influenza + 48 hours or Type B influenza, which are inoculated with first 2 hours, to be started.In A type influenza research, individually or with 10mg/kg Oseltamivir combine The compound (1) of excipient (10mL/kg) and 0.1-10mg/kg dosage are applied in ground, take orally (PO), 2 times a day (BID), continue 10 days.In Type B influenza research, excipient (10mL/kg) and 1- individually or with 10mg/kg Oseltamivir are applied in combination The compound (1) of 10mg/kg dosage, oral (PO) continue 10 days 2 times a day (BID).It weighs to mouse and observes daily 21 days ill signs after infection.In addition, monitoring lung function by unconfined WBP (Buxco, Troy, NY).

A type influenza/PR/8/34 (VR-1469) and Type B influenza/Mass/3/66 (VR-523) derive from ATCC (Manassas, VA).Stock solution is prepared by standard method known in the art.In short, making virus with low infection multiplicity in Madin- Passage in Darby canine kidney cells (mdck cell, CCL-34, ATCC), harvests supernatant after about 48 hours, and with 650x g from The heart 10 minutes.Viral stock solution is frozen in -80 DEG C until the use.In serial dilution viral sample, infection MDCK training After supporting object and measurement induced cytopathic effect (CPE) duplication, the content based on ATP at 96 hours passes through Spearman-Karger Method calculates virus titer (TCID₅₀/ml)(CellTiter-Glo,Promega,Madison WI)。

Using two ways ANOVA and Bonferroni post-hoc tests analysis weight data to compare each group.It will be less than 0.05 P- value is considered as with conspicuousness.

Daily weighing mouse after infection, continues 21 days.Mouse is observed after influenza infection daily, continues 21 days.Think to comment It is divided into following 6 kinds of observations result (> 35%BW mitigation, ruffled fur, posture bow, respiratory distress, activity Reduce or hypothermia) 4 kinds of positive any mouse be it is dying, then implement euthanasia and according to The guideline scoring that VertexInstitutional Animal Care and Use Committee is established is death.It uses Kaplan Meier method analyzes survival rate data.

Mouse is carried out without limitation WBP (Buxco, Troy, NY).Lung function is expressed as to the pause (Penh) of enhancing, i.e., Reflect lung resistance without unit calculated value.The value, which is derived from, changes the holding pressure store that result is floated as animal breath type Change.The bronchoconstriction of animal airway influences air-flow and holding pressure store thus.In expiration (PEP) and air-breathing (PIP) The change of pressure is tracked in the process.Penh value is calculated according to formula Penh=pause x PEP/PIP, is determined wherein " pause " reflects When expiration.So that mouse is adapted to plethysmography room 15 minutes, then acquire data at 1 minute interval, asks flat in 10 minutes Mean value and it is expressed as absolute Penh value.Using two ways ANOVA and Bonferroni post-hoc tests analysis data to compare Each group.P- value less than 0.05 is considered as with conspicuousness.

As a result: evaluation compound (1) combined with Oseltamivir in influenza pulmonary infection mouse model with individual compound (1) or Oseltamivir is compared in the prevention death rate and disease incidence, reduces BW mitigation and prevention and/or restore the energy in lung function Power.The combination does not have adverse effect to the efficiency of every kind of drug compared with showing the drug being administered alone with every kind.It is controlled in addition, combining Treat the failure dosage (compound (1) and Oseltamivir of respectively 0.3 and 10mg/kg of the display as every kind of individual compound (Oselatamivir)) synergistic effect is shown in A type treatment of influenza, merge increased survival rate at this time from 0 and reach 100%. Compound (1) in vivo almost without active (as according to desired by available vitro data) and does not do Type B influenza Disturb the validity of Oseltamivir.

A type influenza mouse model: the control group of whole excipient-treatments is by still illness in the 9th or 10 day.After infection When application in+48 hours, compared with excipient control group, provided with the individual compound of 1,3 and 10mg/kg (1) BID treatment complete Prevention is dead, reduces BW and mitigates and restored lung function (table 17).When starting treatment when+48 is small after A type influenza infection, It can not prevent dead, reduction BW from subtracting with 0.1 and 0.3mg/kg compound (1) and 10mg/kg the Oseltamivir treatment being administered alone Light or recovery lung function.Meaningfully ,+48 hours 0.3mg/kg compound (1) He Aosi being co-administered after A type influenza infection His Wei, which provides, entirely prevents dead, reduction BW to mitigate and restore lung function.

Vivo potency number of the compound (1) of application in+48 hours with or without Oseltamivir after table 17:A type influenza infection According to

Type B influenza mouse model: the control group of whole excipient-treatments still illness when by the 7th or 8 day.Application 1,2h and lasting BID are not provided for 10 days compared with the control group before the individual compound of 3 or 10mg/kg (1)-Type B influenza infection To disease incidence, the significant prevention effect that BW mitigates or lung function is lost.Individual Oseltamivir is applied with 10mg/kg or with 1,3 Or 2h is provided and is prevented completely to dead, reduces BW and mitigate and extensive before 10mg/kg compound (1) combination-Type B influenza infection Lung function (table 18) is answered.

Vivo potency number of the compound (1) of application in+48 hours with or without Oseltamivir after table 18:B type influenza infection According to

Embodiment 8: the in vivoassay that compound (1) is combined with Oseltamivir

From using 5x10³TCID₅₀Start within 24 hours before the A type influenza challenge of A/PR/8/34, with excipient or enhancing dosage The HCl salt 1/2H of horizontal compound (1)₂The form A (the hereinafter referred to as compound (1) of embodiment 8) and zanamivir of O Combined therapy infecting mouse.A type influenza challenge and compound are prepared according to the mode similar with as described in the embodiment 7 (1) suspension.In IN attack and 5x10³TCID₅₀Before A/PR/8/34 24 hours with 0.3mg/kg, 1mg/kg or 3mg/kg IN The mouse of (intranasal) zanamivir treatment under fire, and using 5x10³TCID₅₀A/PR/8/34, which is attacked first 2 hours, to be started, With the mouse of compound (1) treatment in 0.1mg/kg, 0.3mg/kg or 1mg/kg BID 10 days under fire.

Result is summarised in the following table 19 A and 19B.As shown in following table 18A, joined using compound (1) and zanamivir It closes therapy and provides additional survival helpfulness (table 19A).By efficiency quotient, survival rate composite measurement, weight loss and lung Function (% survival rate/(the % weight loss at the 8th day) * (Penh at the 6th day)) is summarised in table 19B.

Table 19A: survival rate: the conjoint therapy of compound (1) and zanamivir

Table 19B: efficiency quotient: the conjoint therapy of compound (1) and zanamivir

Embodiment 9: compound (1) efficiency after the prevention and infection in mouse A type influenza infection model

Material and method

Animal: female 18-20g BALB/c mouse derives from Jackson Laboratories (Bar Harbor, ME) and is used for Antiviral breeding.Animal is maintained on standard rodent chow and can arbitrarily drink tap water.Using it is preceding by they every From 48 hours.

Virus: the A type influenza that mouse adapts to/California/04/2009 (pndH1N1) virus derives from Elena Doctor Govorkova (St.Jude Children ' s Research Hospital, Memphis, TN).By viral stock solution It is expanded in mdck cell, lethality titration is then carried out in BALB/c mouse.A type influenza/Victoria/3/75 (H3N2) Virus derive from American Type Culture collection (American Type Culture Collection) (Manassas, VA).The virus is passed in mouse 7 times so that mouse adapts to it, is then passed on 1 time in mdck cell.It is small in BALB/c The lethality that the virus is further titrated in mouse obtains lethal challenge dosage appropriate.A type influenza/Vietnam/1203/2004 (H5N1) virus derives from Jackie doctor Katz of Center for Disease Control (Centers for Disease Control) (Atlanta,GA).Mouse is set to be exposed to the virus (5MLD50,5PFU/ mouse) of lethal dose, at this dose, elder generation is in 6- Lead to death within 13 days, the death rate is 90-100% when by the 10th day.

Compound: Oseltamivir (as) derive from local pharmacy.When being metabolized in vivo, every Tamiflu Capsule includes 75mg active constituent Oseltamivir carboxylate.Oseltamivir dosage is based on this measured value.The HCl of compound (1) The form A (the hereinafter referred to as compound (1) of embodiment 9) of salt semihydrate is used for this research, and the dosage base of the compound In the HCl salt semihydrate of compound (1).With 0.5% methylcellulose (Sigma, St.Louis, MO) prepare compound (1) Mouse is applied to for oral cavity gavage (p.o.) with Oseltamivir.

Experimental design: intranasally made by intraperitoneal injection ketamine/Xylazine (50/5mg/kg) anesthetized mice and warp Object infects 90- μ l influenza virus suspension.Virus attack is about 4 50% mouse lethal infective doses.As indicated, from virus It attacks first 2 hours or starts to give within 48 hours after attacking and treat, 2 times a day (interval 12 hours), continue 10 days.Feel for evaluating The parameter of dye is survival rate, average number of days of death, weight changes and Lung infection parameter (hemorrhage score, weight and virus titer). In infection every other day to the 21st day each self-weighing animal.Think that mouse dead during first 6 days for the treatment of phase dies of non-influenza It the reason of virus infection and is excluded from total statistical number.

In order to evaluate Lung infection parameter, harvests and (start each group 5 separately set for the purpose from the lung for putting to death animal Animal).It is checked by visual observation from pink to the evaluation empsyxis scoring of the color change of plum color.Such case occurs in lung Regional area, rather than deeper color is altered in steps into entire lung.Hemorrhage score is 0 (normal) to 4 (display plum colors Complete lung) and be thus non-measured value of parameters.Then weighing and is frozen in -80 DEG C at lung.Then, by the lung of thawing in 1ml cell It is homogenized in culture medium, by supernatant flow centrifugation to remove particulate matter, and fluid sample is frozen in again at -80 DEG C.? After preparing the hole 96- mdck cell plate, melt sample, dilutes incremental order dilution with 10- times, and exist by Endpoint Dilution Method Titration, uses 4 micropores/dilution in plate (1).Virus titer is calculated as 50% cell culture infective dose of log10/gram lung It organizes (log10 CCID50/g).

Statistical analysis: the Kaplan-Meir by Mantel-Cox Log-Rank test analysis for multiple-group analysis schemes, so as to Determine significance,statistical.Then, it is examined by Gehan-Breslow-Wilcoxon and carries out paired comparisons.Based on being treated Relative experimental conspicuousness is adjusted the conspicuousness threshold value calibrated to Bonferroni by the quantity of comparison.By kruskal-Wallis test, Then pass through the dead average time of Dunn multiple comparative test analysis and average empsyxis scoring.It is average by ANOVA assessment Weight, lung weight and log10 Pneumovirinae titre, estimate equivalent variability and normal distribution.After ANOVA, pass through Tukey- The more each processing costs of Kramer multiple comparative test.It usesSoftware (GraphPad Software, San Diego, CA) it is analyzed.

As a result it and discusses

The preventive dose response of compound (1) is studied in mouse A type influenza model.2h starts to apply figuration before infection Agent or compound (1), and continue 10 days 2 times a day.Result is summarised in table 20 and 21.Receive the complete of individual excipient Portion mouse by research still illness in the 9th day, and averagely lose~32% weight (BW).With 1,3 or 10mg/kg BID The dose dependent reduction that the compound (1) of application provides complete survival and BW mitigates.It is individually applied with 0.3mg/kg BID Compound (1) provides some survival helpfulnesses (2/8 mouse), and but, there are significant BW to mitigate for mouse.Identical In experiment, 10mg/kg BID Oseltamivir is applied to mouse, for clinical comparable human dose (being based on AUC).All application is difficult to understand The mouse of Si Tawei survives, and there are similar weight loss point compared with the mouse of application 1mg/kg BID compound (1) Cloth.

Compound (1) after infection application in 48 hours when, continuous BID is administered 10 days, it is this using A type influenza/ Effect property (table 22) is still provided in the model of Vietnam/1203/2004 (H5N1) attack.Compound is given with 10mg/kg (1) complete protective effect as shown in Table 20 is provided.

Table 20: using compound (1) and Oseltamivir to A type influenza/California/04/ in BALB/c mouse The prevention effect (prevention) of 2009 (pndH1N1) virus infections

Table 21: compound (1) and Oseltamivir are to A type influenza in BALB/c mouse/Victoria/3/75 (H3N2) virus The effect (prevention) of infection

Table 22:(+48h) using compound (1) and Oseltamivir treat in BALB/c mouse to A type influenza/ The effect of Vietnam/1203/2004 (H5N1) virus infection

Embodiment 10: external efficiency of the compound (1) to the influenza strain of A type spectrum

Cell and virus .Madine Darby dog kidney (MDCK) cell initially derive from American Type Culture collection It (ATCC, Manassas, VA) and is passed on using standard laboratory techniques, is subsequently used for infection measurement.Cell is maintained 37 DEG C supplemented with 10% fetal calf serum (Sigma-Aldrich, St.Louis, MO), 2mM L-Glutamine, 10mM HEPES, The improved Eagle culture medium (DMEM of the Dulbecco of 100U/mL penicillin and 100ug/mL streptomysin (Invitrogen)； Invitrogen, Carlsbad, CA) in.Influenza virus derives from ATCC, Virus Surveillance and Diagnosis Branch of the Influenza Division of the Centers for Disease Control and Prevention(CDC；Atlanta, GA) or Influenza Reagent Resource, Influenza Division, WHO Collaborating Center for Surveillance,Epidemiology and Control of Influenza, CDC.In order to generate viral stock solution, make mdck cell supplemented with 2mM L-Glutamine, 10mM HEPES, 100U/mL Penicillin, 100ug/mL streptomysin and 1 μ g/mL tosyl phenylalanyl chloromethyl ketone (TPCK)-processing tryptose Enzyme (USB Corp.；Santa Clara, CA) DMEM in infect low infection multiplicity (MOI).By cell in 37 DEG C and 5%CO₂ 48h is incubated, hereafter, supernatant is harvested with 900x g centrifugation 10min by using Beckman GS-6R centrifuge.Virus is stored up For solution equal part and it is frozen at -80 DEG C.

Compound is by the free alkali of compound (1) or HCl salt (such as unbodied compound (1) HCl salt, compound (1) the form A of HCl salt semihydrate, amorphous free alkali compound (1)) (the hereinafter referred to as compound of embodiment 10 (1)) it is dissolved in 100% dimethyl sulfoxide (DMSO), the solution of 10mM concentration is made.

Antiviral activity uses CellTiter-Glo (Promega in mdck cell；Madison, WI) such as ATP level The antiviral activity of identified evaluation compound (1).By 50 μ Ls of the mdck cell in black clarification bottom 384- hole plate The upper bed board of VGM to density is 2x10⁴Cell hole.By cell in 37 DEG C, 5%CO₂, under standard humidity be incubated to cell adherence simultaneously And form single layer.After 5h, 40 μ L culture mediums are taken out, and the virus that 15 μ L MOI are 0.005 is added.Using compound as having There are, the 3- times of dilution addition of 25 μ L in the DMEM of complement at 10 points (final DMSO concentration is 0.5%).Internal contrast group is by only wrapping The hole of cell containing cell and untreated virus infection forms.After 72h is incubated, 20 μ LCellTiter-Glo are added to often In a hole and in incubation at room temperature 10min.Use EnVision Multilabel reader (PerkinElmer；Waltham, MA) measurement shines.By using 4- parameter curve method, using Levenburg Marquardt algorithm, (Condoseo is soft Part；Genedata, Basel, Switzerland) it is fitted compound dosage and response data calculating EC₅₀Value (ensures to be uninfected by The compound concentration of 50% cell survival rate in control group).It is taken precautions against in Southern Research Institute in BSL-3 The lower testing in vitro for carrying out hpaiH5N1.

As shown in the following table 23, compound (1) display is to whole effective actives for testing the influenza strains of A type, including comes from H1N1 the and H3N2 reference strain of 1934-2009 and a wide range of popular 2009H1N1 strain A/California/07/2009, A/ Texas/48/2009 and highly pathogenic fowl H5N1 plants of A/VN/1203/2004.Compound (1) is equally effective to all strains, including It is those of resistant to amantadine and neuraminidase inhibitor.It shows to the limited activity of Type B influenza virus.

Table 23: efficiency of the compound (1) to one group of influenza strain

Embodiment 11: the external Collaborative experiment of compound (1) and Oseltamivir, zanamivir or Faville drawing Wei

Combine with neuraminidase inhibitor Oseltamivir carboxylate and zanamivir or polymerase inhibitors T-705 In experiment, the testization in measurements of the mdck cell based on CPE on the 3rd of the infection MOI A/Puerto Rico/8/34 for being 0.01 Object (1) (similarly in the free alkali or HCl salt of the compound (1) in embodiment 10) is closed in 100% dimethyl sulfoxide (DMSO) Solution.Oseltamivir carboxylate and T-705 are dissolved in 100% dimethyl sulfoxide (DMSO)；By zanamivir with the concentration of 10mM It is dissolved in the improved eagle culture medium (DMEM) of Dulbecco and is stored at -20 DEG C.This research uses the independence side Bliss Method (Macsynergy) (such as Prichard, M.N. and C.Shipman, Jr., Antiviral Res, 1990.14 (4-5): ) or Loewe additive property/intermediate effect method (such as Chou, T.C. and P.Talalay, Adv Enzyme p.181-205 Regul, 1984.22:p.27-55).Bliss independence method includes that the inhibitor combination of various concentration is tested in a manner of chessboard, And Loewe independence method includes with the inhibitor combination of the dilution test fixed proportion of different fixed proportions.Also use Compound (1) itself is tested as control, to confirm additive property.Cell survival is measured using CellTiter-Glo Power.

Bliss independence method causes the synergistic effect volume of Oseltamivir carboxylate and zanamivir to be respectively 312 Hes 268；And drawing Wei to obtain synergistic effect volume Faville is 317.Synergistic effect volume is greater than 100 and generally is regarded as cooperateing with work by force With, and the volume of 50-100 is considered as moderate synergistic effect.Loewe additive property method is for Oseltamivir, zanamivir and T- 705 generate 0.58,0.64 and 0.89C.I. (association index) value in the case where 50% effect is horizontal respectively.C.I. value quilt less than 0.8 It is considered as Strong synergy, and the value of 0.8-1.0 is considered as being added to appropriate synergistic effect.These data as shown in Table 24 are total Neuraminidase inhibitor and polymerase inhibitors with enlightenment compound (1) and test have synergistic effect.

Table 24: In Vitro Synergistic Effects and antagonism General description of experiments

Embodiment 12: the efficiency in mouse A type influenza infection model

Compound (1) (amorphous or shape of the semihydrate of compound (1) HCl salt is studied in mouse A type influenza model The preventive dose of formula A (the present embodiment is hereinafter referred to as compound (1)) responds.2h starts to apply excipient or change before infection It closes object (1) and continues 10 days 2 times a day.Receive whole mouse of individual excipient when studying the 9th day still by Infection, and averagely lose~32% its weight (BW).It is provided with the compound (1) of 1,3 or 10mg/kg BID application complete Survival and dose dependent BW mitigate reduce.It is beneficial that some survivals are provided with the compound (1) that 0.3mg/kg BID is applied Property (2/8 mouse), but, there are significant BW to mitigate for mouse.It is difficult to understand to mouse application 10mg/kg BID in identical experiment Si Tawei, for clinical comparable human dose (being based on AUC).All application Oseltamivirs mouse survive, and with application The mouse of 1mg/kg BID compound (1) is compared to there are the distributions of similar weight loss.

Start to apply figuration when by attacking mouse and after infection 24,48,72,96 or 120h with influenza A Agent, Oseltamivir or compound (1), which continue BID, which applies research in 10 days, can postpone compound (1) application and in the model still The degree (table 25) of validity is so provided.Whole excipient control groups are in still illness when by the 8th or 9 day.With excipient Control group comparison entirely prevented when starting application up to 72h after infection with the compound (1) of 1,3 or 10mg/kg BID application Death, and reduce BW mitigation.When only starting application for 24 hours or below after infection with 10mg/kg BID application Oseltamivir Provide complete prevention effect.When postpone again start apply compound when, the compound (1) of 3 or 10mg/kg BID is infecting Complete survival is provided when 96h afterwards and postpones 120h after infection and starts to provide part protective effect when application.

Studying compound (1) is reducing the validity in Pneumovirinae titre.Make mouse infection A type influenza, and for 24 hours Afterwards, excipient, Oseltamivir (10mg/kg BID) or compound (1) (3,10,30mg/kg BID) are applied, until collecting lung simultaneously And viral load (table 26) was measured at the 6th day.Whole compound (1)-administration groups show and apply with Oseltamivir-and excipient- Animal reduces compared to the strong Pneumovirinae titre with significance,statistical.

In order to establish PK/PD model, make mouse infection influenza virus for 24 hours, and then apply compound (1) again for 24 hours.By agent Amount is divided into single dose, 2 dosage or 4 dosage, respectively every 12h or 6h application.Acquisition lung and blood plasma are for measuring tuberculosis Malicious carrying capacity and compound (1) concentration.Each lung Titer Data of these application programs (q6h, q12h and q24h) will be come to each C_max、C_minOr AUC value draws (data are not shown).Although in the reduction of lung titre and C_minBetween there are apparent correlations, still With C_maxAlmost without correlation, and only has weak correlation with AUC.When by the compound measured in blood plasma (1) concentration and measurement Lung titre draw when, with C_minThere are strong correlations.The half maximum of lung titre reduces (2-3log) and appears in close to serum The EC of conversion₉₉(100ng/mL).Similar correlation is being had found between compound (1) concentration in lung titre and the lung of measurement (data are not shown).

The general introduction of survival rate percentage and weight loss percentage in table 25:A type influenza mouse model

^aData come from independent experiment.

^bData come from identical experiment.

^cND, undetermined.

Pneumovirinae titre and Log in table 26:A type influenza mouse model₁₀Reduced general introduction

^aTreatment of animals starts for 24 hours after infection, continues 5 days.

^bPneumovirinae titre is measured when studying the 6th day.

^cND, undetermined.

2 kinds of mode ANOVA and Bonferroni Post are test, P < 0.001 * * *.

Embodiment 13: the influenza challenge of theory verifying

It is used to predict influenza virus medicine having in the natural infection of human body before attenuation influenza challenge model living Effect property (Calfee, D.P., Peng, A.W., Hussey, E.K., Lobo, M.&Hayden F.G.Safety and efficacy of once daily intranasal zanamivir in preventing experimental human influenza A infection.Antivir Ther.4,143-149(1999)；Hayden, F.G. et al. Use of the oral neuraminidase inhibitor oseltamivir inexperimental human influenza.JAMA282, 1240-1246(1999).It is being vaccinated with the healthy will of A type influenza living/Wisconsin/67/2005 (H3N2) attack strain virus The form A (later in the present embodiment referred to as compound (1)) of the HCl salt semihydrate of compound (1) is carried out in hope person Randomization, double blind, placebo-control single centre research.Subject receive 5 daily dosages placebo (N=33) or Compound (1), (QD) (capsule form being made of net compound (1)) one time a day: when on day 1,100mg (N=16), 400mg (N=19) or 900mg, subsequent 600mg 2-5 days (N=20)；Or when on day 1,1200mg, subsequent 600mg 2-5 days (N=18).Subject receives nose swab 3 times a day, and keeps the scoring card in relation to clinical symptoms 3 times a day, 1-7 It, and left from facility at the 8th day, safety follow-up was substantially carried out at the 28th day.It measures (preliminary analysis) and logical Cross the influenza virus of qRT-PCR (secondary analysis) measurement nose swab in cell culture.

Effectiveness analysis is carried out to complete analysis (FA) group, is defined as described complete analysis (FA) group to receive at least one agent All randomized subjects of quantifier elimination drug (compound (1) or placebo) dosage, virus concentration are greater than or equal to TCID when any point-in-time after inoculation in 48h₅₀The lower limit of quantitation of cell culture, or its hemagglutination after seed stage Reaction titre is inhibited to increase 4- times or more (the 1st day) (N=74) from baseline.Security group is inoculated with influenza when being included in the 0th day And receive the placebo of at least one dosage or all subjects (N=104) of compound (1).

Efficiency evaluation

The major measure of this research, which is to be shown in, studies virus reduction between the 1st day (the 1st day medicament administration) to the 7th day AUC dose response trend, as in FA group cell culture measurement in TCID₅₀It is identified.It is average in nose swab AUC virus is observed in reducing with restrictive dose response trend (P=0.036, the Jonckheere- of statistics Terpstra trend test).In addition, carrying out average AUC virus between the placebo collected and each compound (1) dosage group It reduces, averagely matching for the average magnitude of reduction time limit and peak value virus reduction compares (table 27).For 1200/600mg dosage group Observe AUC virus reduction tool significance,statistical reduction (P=0.010, wilcoxon's rank sum test), and for 1200/600mg dosage group, 400mg dosage group and compound (1) dosage group collected, observe that the conspicuousness of peak value reduction subtracts Few (Figure 13).Carry out additional FA group analysis (data are not shown).

The reduction of nose influenza is also quantified by qRT-PCR and result is similar with being observed using cell culture.? Indifference in terms of seroconversion rate of the compound (1) between dosage group and placebo, as anti influenza titre is inoculated with base from preparatory Line starts to increase 4- times or determined above, and the compound (1) that this enlightenment is applied for 24 hours after influenza inoculation does not influence influenza sense It contaminates acquisition rate and can not eliminate then to the humoral immune response of infection (table 28).

Subject records clinical symptoms in diary 3 times a day.Calculate the 1st day to the 7th day clinical AUC and influenza sample disease Shape scoring.It is compared with placebo, the 1200/600mg dosage group of compound (1) is shown in complex clinical symptom option adjusted duration (P= 0.001), the average AUC (P=0.040) of influenza-like symptom and the option adjusted duration of influenza-like symptom aspect have statistically significant Property reduce (P < 0.001) (table 28).

Table 28: average AUC virus reduces, averagely reduces time limit and average peak virus reduction magnitude

AUC: the area under numerical value and time graph；CI: confidence interval；NA: not applicable；QRT-PCR: quantitative reverse transcription is poly- Synthase chain reaction；SD: standard deviation；TCID50:50% tissue culture infection.

Note: significance,statistical P value (P < 0.05) is indicated with boldface type.

^aP=0.036, for the dose response trend of the AUC from Jonckheere-Terpstra trend test.

^bP value is calculated according to wilcoxon's rank sum test.

^cP value is calculated according to ANOVA.

^dP value is calculated according to Log-Rank test.

^eP=0.031, for the dose response trend of the AUC from Jonckherre-Terpstra trend test.

^fSerum-conversion ratio is defined as in the follow-up compared with the limit anti influenza antibody titer >=4- times and increases.P value makes With the definite checking computation of Fisher.

Table 28: the average AUC of complex clinical symptom and influenza-like symptom, option adjusted duration and peak averaging magnitude

AUC: the area under numerical value and time graph；CI: confidence interval；NA: not applicable.

Note: significance,statistical P value (P < 0.05) is indicated with boldface letter.

^bP value is calculated according to wilcoxon's rank sum test.

^cP value is calculated according to ANOVA.

^dP value is calculated according to Log-Rank test.

Safety evaluatio

Compound (1) be substantially resistant to it is receiving and there is no because compound (1)-related reactions (AE) caused by interruption, Also without any serious adverse reaction.It presents in any treatment group and the column of adverse reaction occurs in >=10% subject Table (table 29).Influenza-like illness be the adverse reaction of most frequent report and by placebo and compound (1) group close to equal proportion Subjects reported.The adverse reaction of >=10% incidence difference occurs between group and placebo recipients for compound (1) are as follows: Serum phosphorus levels reduction (18.1%, compound (1)；0%, placebo), rhinorrhea (compound (1), 4.2%；18.8%, placebo) With nasal congestion (1.4%, compound (1)；15.6%, placebo).In addition, being observed in placebo and compound (1) recipient It is increased to alanine aminotransferase (ALT).Reach 1600mg in single dose and multi-dose reaches daily 800mg change in 10 days The internal dosage of people for the first time for closing object (1) rises in proportion in research, and dysfunction of liver is both not observed, blood is also not observed Clear phosphate decline；Previous utilization upper respiratory tract infection reports ALT and increases and serum phosphate reduction.

Table 29: in any treatment group >=10% subject in adverse reaction list occurs

Note: once then statistics has the subject of a variety of adverse reactions in AE.Subject is likely to occur multiple classes Type.

^aThe single loading dose of 900mg at the 1st day and 600mg qd at the 2-5 days.

^bThe single loading dose of 1200mg at the 1st day and 600mg qd at the 2-5 days.

^cInfluenza-like illness, as determined by effectiveness analysis, based on the parameter evaluation listed in text.Influenza-like illness AE determined by clinician.

It discusses

In the influenza challenge research carried out in healthy volunteer, compound (1) passes through TCID₅₀Cell culture and qRT- PCR shows the dose response trend of the AUC virus titer in nose swab, and the compound (1) for the maximum dose level evaluated leads to AUC Virus titer and AUC and flu symptom time limit are remarkably decreased.Although not seen in the second maximum dose level group 900/600mg (table 27) The similar improvement magnitude more than placebo is observed, but the dosage is shown really with 1200/600mg dosage in complex clinical symptom The result that (table 28) is similar in terms of the average AUC of influenza-like symptom terminal；Complete reason is not yet received in the reason of this deviation Solution.Although POC test in specific safety trend is encountered, it has been observed that phosphate reduce and ALT increase enlightenment The appropriate monitoring needs of two kinds of parameters are for following research.

In short, the limitation of influenza challenge model is that the influenza virus for this research is the strain especially selected, so as not to Generate the most serious clinical symptoms of influenza infection.In addition, the virus inoculation object of application is likely larger than native influenza exposure.Cruelly Timing application compound (1) for 24 hours may be not the time limit of reality for the therapy that starting community starts after dew, and wherein patient is logical Chang Buhui seek diagnose or treat, until conspicuousness symptom occurs in they, it may be possible to after exposure for 24 hours more than.However, by Be seeded initially remote low virus titer in the subject of natural infection, thus time scale and it is indirect be it is comparable compared with.

Briefly, compound (1) is effective A type influenza PB2 inhibitor, and which represent the anti-of brilliant and new type Viral agent.The characteristic of this inhibitor as described in preclinical and clinical data shows that compound (1) is further evaluated Encouraging candidate, several potential advantages therein have been more than the current antivirotic for treating influenza infection.

All references provided herein are fully incorporated herein by reference.All abbreviations, symbol and used as used herein Example uses those consistent with contemporary scientific literature.See, for example, Janet S.Dodd etc., The ACS Style Guide:A Manual for Authors and Editors, second edition, Washington, D.C.:American Chemical Society, 1997。

Embodiment 14: compound (1) rich in deuterium

The compound (1) of deuterium is rich according to the synthesis of following scheme 1:

Scheme 1:

Reagent and condition: (step 14a) maleic anhydride, CHCl₃；(step 14b)

Betaquinine, ethyl alcohol, toluene；(step 14c) DPPA, Et₃N,90℃,BnOH；(step 14d) H₂,Pd/C,MeOH； (step 14e) amine 14-5,ⁱPr₂NEt,THF,70℃；(step 14f) HCl, dioxanes, MeCN, 80 DEG C；(step 14g) NaOH, THF,MeOH.

14A: compound (14-1)

Potassium tert-butoxide (9.663g, 86.11mmol) is dissolved in DMSO-d₆(30.00mL), is put into nitrogen atmosphere.It is added Hexamethylene-solution of the Isosorbide-5-Nitrae-diene (6g, 74.88mmol) in pentane (60.00mL), which is stirred in nitrogen atmosphere 2.5hrs.Take out DMSO-d₆Layer, is added fresh 30mL DMSO-d₆With potassium tert-butoxide (9.663g, 86.11mmol).Lasting stirring Overnight.Each layer is separated, D is used₂O (50mL) washs pentane layer, uses Na₂SO₄It is dry, generate 1,2,3,4,5,5,6,6- eight deuterated rings Hex- 1,3- diene (14-1), as solution in next step.The mixture of reaction generation 1,3- and 1,4- diene isomerism body. Only 1,3- diene participates in subsequent reaction step.

14b:3a, 4,7,7a- tetrahydro -4,7- ethano- isobenzofuran -1,3- diketone -4,5,6,7,8,8,9,9-d₈ (14-2)

With chloroform (50mL) dilution the deuterated hexamethylene -1,3- diene (14-1) of 1,2,3,4,5,5,6,6- eight (6.5g, Pentane solution 74.0mmol) is handled with maleic anhydride (8.0g, 81.4mmol).The reaction mixture was stirred at room temperature Night.Solvent, the semi-solid residue handled with MeOH is evaporated under reduced pressure.After ten minutes, MeOH slurry is cooled to about for stirring 20℃.By filtering the precipitating collected, is washed with 3 fractions (5mL) cold methanol, obtain product (14-2), admittedly for white Body:¹H NMR analyzes (CDCl₃) 3.15 (s, 2H) show net product and the incorporation of 95% deuterium.

14c:(+/-)-trans- bicyclic [2.2.2] the octyl- 5- alkene -2- carboxylic -1,4,5,6,7,7,8,8-d of -3- (carbethoxyl group)₈ Sour (14-3)

To 3- neck RBF connection addition funnel and internal temperature probe in nitrogen atmosphere.It is packed into 3a into flask, 4,7, 7a- tetrahydro -4,7- ethano- isobenzofuran -1,3- diketone -4,5,6,7,8,8,9,9-d₈(14-2)(2.68g, 14.39mmol), betaquinine (5.24g, 15.83mmol) and dry toluene (40mL).Magnetic stirs the reaction system, is cooled to -25 DEG C (cold finger cooling).The solution of dehydrated alcohol (8.40mL, 143.90mmol) in dry toluene (13.4mL) is added, lasts 25 Minute, maintain internal temperature to be lower than -25 DEG C.The reaction mixture is stirred overnight at about -20 DEG C.Pass through filtering acquisition precipitating Gel sample solid is washed with toluene (3 × 30mL), is dissolved in 1N HCl/EtOAc aqueous solution (300mL 1:1 mixture).Stirring is double Phase mixture, until all precipitating dissolutions.Each layer is separated, washs organic layer with water (2 × 100mL), salt water (100mL), is used Na₂SO₄It dries, filters, is concentrated with rotary evaporator low temperature, obtains the desired product of 800mg (14-3), without further purification It uses.

14d:(+/-)-trans-Ethyl -3- (((benzyloxy) carbonyl) amino) bicyclic [2.2.2] octyl- 5- alkene -2- carboxylic acid Ester -1,4,5,6,7,7,8,8-d₈(14-4)

To bicyclic [2.2.2] the octyl- 5- alkene -2- carboxylic -1,4,5,6,7,7,8,8-d of (+/-)-trans- -3- (carbethoxyl group)₈Acid (14-3) (0.60g, 2.58mmol) in the solution in toluene (4.5mL) be added diphenyl phosphoryl azide (0.81g, 0.63mL, 2.84mmol), triethylamine (0.40mL, 2.84mmol) then is added.The reaction mixture is heated to 90 DEG C 2 small When.Benzylalcohol (0.35mL, 3.34mmol) is added into the mixture, is heated overnight at 90 DEG C.The reaction mixture is cooled to Room temperature is dispensed into EtOAc and saturation NaHCO₃Aqueous solution.Each layer is separated, with saturation NH₄Cl aqueous solution, salt water washing organic phase, Use Na₂SO₄It dries, filters, is evaporated to dryness.It is purified by silica gel chromatography thick residue (0-35-100%EtOAc/ hexane-use CAMA dyeing).¹H NMR is shown desired product (14-4), and still has benzylalcohol impurity.Without further purification by substance For in next step.

14e:(+/-) bicyclic [2.2.2] octane -2- carboxylate -1,4,5,5,6,6,7,8-d of-trans-Ethyl -3- amino₈ (14-5)

Palladium (0.052g, 0.049mmol) is fitted into hydrogenation vessel (in nitrogen atmosphere), it is wet with about 5mL methanol.To this In suspension be added (+/-)-it is trans--(2S, 3S) -3- (((benzyloxy) carbonyl)-amino) bicyclic [2.2.2] octyl- 5- alkene -2- carboxylic Acetoacetic ester -1,4,5,6,7,7,8,8-d₈The solution of (14-4) (0.521g, 1.547mmol) in methanol (20mL).This is anti- Mixture is answered to be hydrogenated (44PSI) overnight.Pressure is emptied, catalyst is filtered out.Whole volatile materials are removed in vacuum.It will be thick Product (14-5) uses without further purification.

14f:(+/-)-trans-Ethyl -3- ((the fluoro- 2- of 5- (fluoro- 1- tosyl -1H- pyrrolo- [2,3-b] pyrrole of 5- Pyridine -3- base) pyrimidine-4-yl) amino) bicyclic [2.2.2] octane -2- formic acid esters -1,4,5,5,6,6,7,8-d₈(14-7)

To (+/-)-it is trans--carboxylic acid, ethyl ester -1,4,5,5,6,6,7 (2S, 3S) -3- amino bicyclic [2.2.2] octane -2-, 8-d₈(14-5) (0.317g, 1.547mmol) and the fluoro- 3- of 5- (the fluoro- 4- methylsulfinyl-pyrimidine -2-base of 5-) -1- (p- first Phenyl sulfonyl) pyrrolo- [2,3-b] pyridine (14-6) (0.694g, 1.547mmol) in the suspension in THF (10mL) plus Enter n,N-diisopropylethylamine (0.808mL, 4.641mmol), which is heated to 70 DEG C overnight.With EtOAc and Water dilutes the reaction mixture.Each layer is separated, organic phase, dry (MgSO are washed with brine₄), it filters, vacuum concentration.Pass through silicon Glue chromatography purification of crude product (14-7) (0-100%EtOAc/ hexane), obtains desired product.

14g:(+/-)-trans-Ethyl -3- ((the fluoro- 2- of 5- (fluoro- 1H- pyrrolo- [2,3-b] pyridin-3-yl of 5-) pyrimidine - 4- yl) amino) bicyclic [2.2.2] octane -2- formic acid esters -1,4,5,5,6,6,7,8-d₈(14-8)

To (+/-)-it is trans--(2S, 3S) -3- ((the fluoro- 2- of 5- (fluoro- 1- tosyl -1H- pyrrolo- [2,3-b] pyrrole of 5- Pyridine -3- base) pyrimidine-4-yl) amino) bicyclic [2.2.2] octane -2- Ethyl formate -1,4,5,5,6,6,7,8-d₈(14-7) (373mg, 0.6325mmol) in the solution in acetonitrile (6mL) be added HCl (dioxane of 800 μ L 4M, 3.200mmol).The reaction mixture is stirred at room temperature 2 hours.Then the reaction mixture is heated to 80 DEG C of 6hrs, so After be cooled to room temperature, be stirred overnight.LC/MS is not analysis shows that reaction completes.6ml CH is added into the mixture again₃CN and 800 μ l 4N HCl/ dioxanes.The reaction mixture is heated to 80 DEG C 4 hours.Whole volatility objects are removed under reduced pressure Matter.With EtOAc, saturation NaHCO₃Aqueous solution dilutes residue.Each layer is separated, organic phase is washed with brine, uses MgSO₄It is dry, mistake Filter, vacuum concentration.It is purified by silica gel chromatography thick residue (0-100%EtOAc/ hexane), obtains desired product (14- 8):¹H NMR (300MHz, d6-DMSO) δ 12.28 (s, 1H), 8.50 (dd, J=9.8,2.8Hz, 1H), 8.23 (ddd, J= 12.6,6.2,2.7Hz, 2H), 7.60 (d, J=6.9Hz, 1H), 4.73 (t, J=6.5Hz, 1H), 4.30-3.85 (m, 2H), 2.89 (d, J=6.8Hz, 1H), 1.59-0.96 (m, 4H)

14h:(+/-)-trans-Ethyl -3- ((the fluoro- 2- of 5- (fluoro- 1H- pyrrolo- [2,3-b] pyridin-3-yl of 5-) pyrimidine - 4- yl) amino) bicyclic [2.2.2] octane -2- first -1,4,5,5,6,6,7,8-d₈Sour (1)

To (+/-)-trans-Ethyl -3- ((the fluoro- 2- of 5- (fluoro- 1H- pyrrolo- [2,3-b] pyridin-3-yl of 5-) pyrimidine -4- Base) amino) bicyclic [2.2.2] octane -2- formic acid esters -1,4,5,5,6,6,7,8-d₈(14-8) (0.165g, 0.379mmol) is molten NaOH (1mL 2M solution, 2.000mmol) is added in solution in THF (3.0mL) and methanol (1mL), which is mixed Object is stirred at room temperature 3 hours.LC/MS is not analysis shows that reaction completes.The reaction mixture is warmed to 45 DEG C 2 hours, then It is warmed to 55 DEG C 30 minutes.The reaction mixture is diluted into saturation NH₄Cl aqueous solution.A few drop 1N HCl are added with by pH tune It is whole to about 6.5.Product is extracted with EtOAc.Use MgSO₄Dry organic phase, is filtered, and vacuum concentration obtains desired product (1) (97.5% purity is measured by NMR, LC/MS and HPLC):¹H NMR (300MHz, d6-DMSO) δ 12.30 (d, J=14.2Hz, 2H), 8.79-7.94 (m, 4H), 7.58 (s, 1H), 4.68 (s, 1H), 2.84 (s, 1H), 1.85 (d, J=85.0Hz, 1H), 1.58-1.05(m,2H).

Embodiment 15: compound (1) rich in deuterium

Alternatively, the compound (1) for being rich in deuterium can be synthesized according to following scheme 2:

Scheme 2:

Other embodiments

Although above description is intended to illustrate to be not intended to limit it will be appreciated that describing the present invention together with its detailed description The scope of the present invention, but limited by scope of the appended claims.Other aspects, advantage and modification are in following following claims Within the scope of.

Claims (30)

1. the polymorphic of compound (1) or its pharmaceutically acceptable salt, wherein compound (1) is indicated by following structural formula:

And wherein the polymorphic is selected from:

The HCl salt 1/2H of compound (1)₂The form A of O；

The HCl salt 3H of compound (1)₂The form D of O；

The form D of the HCl salt of compound (1)；

The form A of compound (1)；With

The form A of the toluene fulfonate of compound (1).

2. the polymorphic of claim 1, wherein the polymorphic is the HCl salt 3H of compound (1)₂The form D of O.

3. the polymorphic of claim 2, the wherein HCl salt 3H of compound (1)₂The form D of O is characterized in that being equivalent in X- To spend 7.1 ± 0.2,11.9 ± 0.2,19.2 ± 0.2 and 12.4 ± 0.2 2- θ value measured in powder diffraction pattern One or more peaks.

4. the polymorphic of claim 2, the wherein HCl salt 3H of compound (1)₂The form D of O is characterized in that being equivalent in C¹³ 20.7 ± 0.3ppm, 27.4 ± 0.3ppm, 104.8 ± 0.3ppm, 142.5 ± 0.3ppm and 178.6 in SSNMR spectrum ± One or more peaks of 0.3ppm.

5. the polymorphic of claim 1, wherein the polymorphic is the form D of the HCl salt of compound (1).

6. the polymorphic of claim 5, wherein the form D of the HCl salt of compound (1) is characterized in that being equivalent in X-ray powder One or more peaks of 5.8 ± 0.2,19.5 ± 0.2 and 17.1 ± 0.2 2- θ value of measurement are spent in last diffraction pattern.

7. the polymorphic of any one of claim 6, wherein the form D of the HCl salt of compound (1) is characterized in that being equivalent in C¹³ 29.4 ± 0.3ppm, 53.4 ± 0.3ppm, 113.3 ± 0.3ppm, 135.4 ± 0.3ppm and 177.8 in SSNMR spectrum ± One or more peaks of 0.3ppm.

8. the polymorphic of claim 1, wherein the polymorphic is the form A of compound (1).

9. the polymorphic of claim 8, wherein the form A of compound (1) is characterized in that being equivalent in X-ray powder diffraction One or more peaks of 15.5 ± 0.2,18.9 ± 0.2 and 22.0 ± 0.2 2- θ value of measurement are spent in pattern.

10. the polymorphic of claim 9, wherein the form A of compound (1) is characterized in that being equivalent in C¹³In SSNMR chromatography 21.0 ± 0.3ppm, 28.5 ± 0.3ppm, 50.4 ± 0.3ppm, 120.8 ± 0.3ppm, 138.5 ± 0.3ppm, and 176.2 One or more peaks of ± 0.3ppm.

11. the polymorphic of claim 1, wherein the polymorphic is the form A of the toluene fulfonate of compound (1).

12. the polymorphic of claim 11, wherein the form A of the toluene fulfonate of compound (1) is characterized in that being equivalent to In X-ray powder diffraction figure case with spend measurement 7.2 ± 0.2,9.3 ± 0.2,13.7 ± 0.2,14.3 ± 0.2,14.7 ± 0.2, one or more peaks of 16.9 ± 0.2,18.7 ± 0.2,26.3 ± 0.2 and 26.9 ± 0.2 2- θ value.

13. pharmaceutical composition, the polymorphic comprising claim 1 and at least one pharmaceutically acceptable carrier or excipient.

14. reduce external biological sample in or subject in influenza virus amount or inhibit influenza virus duplication method, packet Include the polymorphic of the compound (1) of any one of claim 1-12 a effective amount of to the sample administration.

15. inhibiting the method that in external biological sample or influenza virus is replicated in subject, including effective to the sample administration The polymorphic of the compound (1) of any one of the claim 1-12 of amount.

16. the polymorphic of the compound (1) of any one of claim 1-12 is preparing the drug for treating the influenza of subject In purposes.

17. the purposes of claim 16, wherein the drug further includes one or more other therapeutic agents.

18. the purposes of claim 17, wherein the other therapeutic agent includes antiviral drugs.

19. the purposes of claim 18 is selected from Oseltamivir or bundle wherein the antiviral drugs is neuraminidase inhibitor Na meter Wei.

20. the purposes of claim 18 draws Wei selected from Faville wherein the antiviral drugs is polymerase inhibitors (flavipiravir)。

21. the HCl salt 3H of prepare compound (1)₂The method of the form D of O, wherein compound 1 is indicated by following structural formula:

This method comprises:

(a) HCl and compound (1) are mixed in the solvent system comprising water, are equal to or more than wherein the solvent system has 0.9 water activity；Or

(b) the HCl salt 1/2H of compound (1) is stirred in the solvent system comprising water₂The form A of O, wherein the solvent system Uniting has the water activity for being equal to or more than 0.9.

22. the method for claim 21, wherein the solvent system also includes one or more organic solvents, selected from chlorobenzene, Hexamethylene, 1,2- dichloroethylene, methylene chloride, 1,2- dimethoxy-ethane, DMAC N,N' dimethyl acetamide, N, N- dimethyl formyl Amine, 1,4- dioxanes, cellosolvo, formamide, hexane, 2-methyl cellosolve, methyl butyl ketone, hexahydrotoluene, N- Methyl pyrrolidone, nitromethane, pyridine, sulfolane, tetrahydrofuran (THF), naphthane, toluene, 1,1,2- trichloro ethylene and Dimethylbenzene, acetic acid, acetone, anisole, n-butyl alcohol, 2- butanol, butyl acetate, t-butyl methyl ether, cumene, heptane, acetic acid are different Butyl ester, isopropyl acetate, methyl acetate, 3- methyl-1-butanol, methyl ethyl ketone, methyl iso-butyl ketone (MIBK), 2- methyl-1-propyl alcohol, Ethyl acetate, ether, Ethyl formate, pentane, 1- amylalcohol, 1- propyl alcohol, 2- propyl alcohol, propyl acetate or its arbitrary combination.

23. the method for claim 21, wherein the solvent system also includes one or more organic solvents, selected from chlorobenzene, Hexamethylene, 1,2- dichloroethanes, methylene chloride, 1,2- dimethoxy-ethane, formamide, hexane, 2-methyl cellosolve, methyl fourth Base ketone, hexahydrotoluene, nitromethane, naphthane, dimethylbenzene, toluene, 1,1,2- trichloroethanes, acetone, anisole, 1- fourth Alcohol, 2- butanol, butyl acetate, t-butyl methyl ether, cumene, ethyl alcohol, ethyl acetate, ether, Ethyl formate, heptane, acetic acid are different Butyl ester, isopropyl acetate, methyl acetate, 3- methyl-1-butanol, methyl ethyl ketone, 2- methyl-1-propyl alcohol, pentane, 1- propyl alcohol, 1- amylalcohol, 2- propyl alcohol, propyl acetate, tetrahydrofuran or methyltetrahydrofuran.

24. the method for claim 21 is selected from iso- third wherein the solvent system also includes one or more organic solvents Alcohol, acetone or its arbitrary combination.

25. the method for the form D of the HCl salt of prepare compound (1), wherein compound (1) is indicated by following structural formula:

This method comprises:

Make the HCl salt 1/2H of compound (1)₂The form A of O is dehydrated.

26. the method for the form A of prepare compound (1), wherein compound (1) is indicated by following structural formula:

This method comprises:

(a) solvent that unbodied compound (1) or compound (1) are stirred in the solvent system comprising water and ethyl alcohol closes Object.

27. the method for the form A of the toluene fulfonate of prepare compound (1), wherein compound (1) is indicated by following structural formula:

This method comprises:

Stir the mixture of following substance: the solvate of amorphous compound (1) or compound (1), p- toluenesulfonic acid and packet Solvent system containing acetonitrile.

28. the 2- methyl-tetrahydrofuran solvent of compound (1) closes object, wherein compound (1) is indicated by following structural formula:

29. the compound (1) of any one of claim 1-12 or the polymorphic of its pharmaceutically acceptable salt are in preparation for controlling The purposes in the drug of influenza infection is treated, the drug is prepared to such form: polymorphic therein is with 100mg- 1,600mg dosage is applied to subject, wherein the dosage daily administration 1 time, 2 times or 3 times.

30. the purposes of claim 29, the drug is prepared to such form: applying when wherein on day 1 to subject The loading dose of 600mg-1,600mg, and 400mg-1, the dosage of 200mg are applied to subject in remaining treatment time limit.